bluuebunny/crossref_metadata_2025 huggingface dataset:
OT3-01-07: The BEACON Study (BrEAst Cancer Outcomes with NKTR-102): A Phase 3 Open-Label, Randomized, Multicenter Study of NKTR-102 Versus Treatment of Physician's Choice (TPC) in Patients (pts) with Locally Recurrent or Metastatic Breast Cancer (MBC) Previously Treated with an Anthracycline, a Taxane, and Capecitabine (ATC).
Authors: Awada A , Leung ACF , Zhao C , Hannah AL , Perez EA
Published: June 2012

Background

NKTR-102 is a next-generation topoisomerase I inhibitor-polymer conjugate with a markedly reduced Cmax and a continuous exposure profile compared to irinotecan. A phase 2 trial of single-agent NKTR-102 compared a dose of 145 mg/m2 every 2w or every 3w in 3rd-line MBC (Awada et al, ASCO 2011). Overall the ORR was 29% (including 3% CR) with the prior ATC subset demonstrating an ORR of 31%. Dosing every 3w was better tolerated; in this arm, median PFS equaled 5.3m and median OS equaled 13.1m.

Trial Design: NKTR-102 will be compared to TPC in an open-label, randomized, parallel, two arm multicenter Phase 3 pivotal study in pts with previously treated locally recurrent or metastatic breast cancer.

Key Entry Criteria: Adult females, with ECOG 0 or 1 with adequate liver, kidney and marrow function. All patients must have received prior therapy with an anthracycline (in neo/adjuvant or metastatic setting or both), a taxane (in neo/adjuvant or metastatic setting or both) and capecitabine (in neo/adjuvant or locally advanced/metastatic setting or both) unless not medically appropriate or explicitly contraindicated for the patient. All chemotherapy- and radiation-related toxicities must have resolved to ≤ Grade 1, except for stable sensory neuropathy ≤ Grade 2 and alopecia. Pts with brain metastases may be eligible, if stable for prior 4 weeks without steroids. Pts with Grade ≥ 2 pre-existing diarrhea or receiving chronic anti-diarrheal supportive care are not eligible.

Statistical Methods: The primary efficacy endpoint is OS. Secondary endpoints include ORR by RECIST v1.1 and PFS. Pts will be randomized 1:1 to NKTR-102 given IV at 145 mg/m2 over 90-min every 21 days or TPC. Patients randomized to TPC will receive single agent chemotherapy, limited to choice of one of the following 5 agents: eribulin, ixabepilone, vinorelbine, gemcitabine, or a taxane. The investigator must decide which agent will be given to the patient prior to randomization. Pts will be stratified by geographical region and disease characteristics.

Target Accrual: Approximately 840 patients (420 pts per treatment arm) will be randomized in order to obtain 615 deaths (alpha = 0.05; power 90% with one interim analysis scheduled when 50% of the deaths have occurred). PK sampling will be performed in a subset of pts and blood and/or tumor samples are planned for potential predictive markers of response and toxicity. Enrollment is expected to remain open until the end of 2013.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT3-01-07.
P1-01-03: The Cytotoxicity of Select Neutrophils in Cancer Patients and the Role of Chemokines in Inducing Neutrophil Cytotoxicity.
Authors: Comen E , Granot Z , Norton L , Benezra R
Published: June 2012

Background: Using murine mammary tumor models, recent research conducted by our laboratory at the Sloan-Kettering Institute indicates that select neutrophils are mobilized by a primary breast tumor and uniquely have the capacity to inhibit metastatic seeding in the lung (Granot Z et al. unpublished). We sought to evaluate the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with those from women without breast cancer and women with DCIS. In addition, we sought to determine whether the addition of select chemokines to neutrophils from a DCIS patient could induce cytotoxicity.

Methods: Neutrophils were purified from the blood of 21 newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, 9 healthy female volunteers with no history of any cancer, and 3 patients with newly diagnosed DCIS. Cytotoxicity was evaluated by incubating isolated neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity, as a reflection of% cell kill, was measured using a Bio-Tek microplate luminescence reader. Neutrophils from a DCIS patient with low cytotoxicity were then co-cultured with various CC chemokines (CCl2, CCL3 and CCL5) or CXCL chemokines (CXCL1, CXCL12, and CXCL16) at 100ng/ml.

Results: Significant cytotoxicity was notably observed when MDA-MB-231 cells were co-cultured with neutrophils purified from patients with invasive tumors. Pre-operative breast cancer patients (n=21) had a cell kill range of 0–30% (mean = 12.1%), whereas healthy subjects (n=9) had a cell kill range of 0.2-8% (mean = 2.6%), p<0.004. DCIS patients (N=3) had a cell kill range of 3–4% (mean = 2.7). The addition of select chemokines to neutrophils from a DCIS patient with low cytotoxicity (3.2%) resulted in significant increases in cytotoxicity. Table 1 indicates the relative cytotoxicity percentages from the addition of each chemokine.

Conclusions: To date, this preliminary work is the first to demonstrate the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with those from women without breast cancer. We further demonstrate the novel induction of neutrophil cytotoxicity by select chemokines. Further studies are needed to evaluate the prognostic and therapeutic role of cytotoxic neutrophils as well as the role of chemokines in neutrophil cytotoxicity.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-03.
P1-01-04: Immunological Effects of Bisphosphonates on gdT Cells in Breast Cancer.
Authors: Sugie T , Tanaka Y , Toi M , Minato N
Published: June 2012

Background: Human γδT cells play an important role in the first-line of defence. Accumulating data demonstrate that γδ T cells recognize nonpeptide antigens such as IPP and HMBPP and activated γδT cells have potent cytotoxic activity against tumor cells. Nitrogen-containing bisphosphonates like zoledronic acid inhibit farnesyl diphosphate synthetase, resulting in the accumulation of upstream metabolites such as IPP. Previous studies on the adoptive transfer of ex-vivo amplified γδ T cells demonstrated the safety and feasibility of γδT cell-based immunotherapy for metastatic cancer. According to ABCSG-12 clinical trial, zoldedoric acid improved DFS of patients with early-stage breast cancer under adjuvant endocrine therapy. Whereas a precise mechanism remained unclear, it is likely that γδT cells are involved in this anti-tumor effect in breast cancer.

Materials and Methods: Peripheral blood mononuclear cells derived from 63 (54 untreated non-metastatic and 9 metastatic) patients with breast cancer were examined for the baseline population of γδT cells and their proliferative responses to zoledronic acid in vitro. Tumor-infiltrating lymphocytes were isolated from 20 tumor biopsies and 2 pleural effusions. Peripheral blood γδT cells were stimulated with 5 μM zoledronic acid and cultured in the presence of 100 IU/ml IL-2 for 10 days. Amplified γδ T cells were analyzed for cytokine production and cytotoxic activity.

Results: The proportion of γδ (TCRVδ2 -bearing) T cells among peripheral blood CD3+ cells was 4.2 ± 5.0%. Although it was equivalent to the previously reported value of 4.3 ± 2.2% in healthy volunteers, 14% (9/63) of patients exhibited an increase in the number of peripheral blood γδT cells at baseline. The level of TCRVδ2 T cells with potent anti-tumor activity was significantly higher than that of TCRVδ1 T cells (4.2± 5.0% vs. 1.5 ± 1.8%, p<0.001), whereas they were present in tumor-infiltrating cells at essentially the same levels (4.4± 9.6% vs. 3.7 ± 9.7%, p=0.434). Percentage of TCR Vδ2+ CD3+ cells among amplified γδ T cells in vitro and cytokine (IFN-γ and TNF-α) production by them were significantly proportional to the initial level of TCRVδ2+/CD3+ ratio in peripheral blood. Upon treatment with zoledronic acid, breast cancer cell lines became susceptible to γδ T cells.

Conclusions: γδ (TCR Vδ2-bearing) T cells increased in some patients with breast cancer and the TCRVδ2+/CD3+ ratio at baseline was correlated with the efficiency of proliferation and cytotoxic activity when treated with zoledronic acid. Zoledronic acid efficiently sensitized breast tumor cells to be lyzed by γδ T cells. The present results suggest that γδ T cells stimulated with zoledronic acid can be utilized for immunotherapy against breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-04.
P1-01-11: CD4+CD25highCD127low/− Regulatory T Cells Have Immunosuppressive Function in Patients with Breast Cancer.
Authors: Sakemura N , Nakatsugawa M , Ito H , Isaka H , Imi K , Tazaki E , Miyamoto K , Wada N , Imoto S , Nakatsura T
Published: June 2012

CD4+CD25highFoxp3+ regulatory T cells play a central role in self-tolerance and suppress antitumor immune response. Recent studies have shown that low levels of the IL-7 receptor (CD127) are expressed on CD4+CD25highFoxp3+ regulatory T cells surfaces and the expression of CD127 is inversely correlated with the suppressive function of CD4+CD25high regulatory T cells. We evaluated the frequency of CD4+CD25highCD127low/− T cells in peripheral blood lymphocytes of 72 breast cancer patients and 21 healthy volunteers. The expression of Foxp3 mRNA was inversely correlated with the CD127 expression and correlated with CD25 expression. The frequency of CD4+CD25highCD127low/− T cells in breast cancer patients was significantly higher than that of healthy volunteers (p=0.0045). CD4+CD25highCD127low/− T cells were increased in hormone receptor negative patients and HER2 positive patients, but no statistical significant was observed in stage progression. In addition, the frequency of CD4+CD25highCD127low/− T cells decreased after curative resection in breast cancer patients (p=0.005). CD4+CD25highCD127low/−T cells also suppress proliferation of autologous CD4+CD25− helper T cells and CD8+ cytotoxic T cells in CFSE-based cell proliferation assay. These findings suggest that CD4+CD25highCD127low/− T cells a useful marker for regulatory T cells in breast cancer. Circulative peripheral regulatory T cells may participate in immune tolerance to breast carcinoma.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-11.
P1-02-06: Silencing of IGF-1R Has Paradoxical Effects in Triple Negative Breast Cancer Phenotypes.
Authors: Taliaferro-Smith LD , Oberlick EM , Liu T , Eggers C , Kline ER , Nagaraju GPC , Marcus A , O'Regan R
Published: June 2012

Background: Triple negative (TN) breast cancers are a heterogeneous group of breast cancers with a poor prognosis in part due to a lack of effective targeted agents. Insulin-like growth factor-1 receptor (IGF-1R) has been shown to play a role in breast cancer cell proliferation, adhesion, invasion, and migration and is overexpressed in more than a third of TN breast tumors. We hypothesized that IGF-1R could be a therapeutic target for a subset of TN breast cancers. Methods: We evaluated the effects of IGF-1R silencing on the metastatic properties of TN breast cancer cells by knock down in two morphologically distinct TN breast cancer cell lines using shRNA lentiviral techniques. Reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting were used to detect mRNA and protein expression levels, respectively, of IGF-1R signaling molecules. Anchorage-dependent growth and Matrigel chamber assays were performed to assess the effects of IGF-1R silencing on colony formation and invasion of TN breast cancer cells, respectively; wound-healing and spheroid migration were also performed to assess the effects of IGF-1R inhibition on TN breast cancer cell motility. Results: Stably transfected mesenchymal MDA-MB-231 TN cells showed effective downregulation of IGF-1R protein expression, which resulted in mesenchymal-to-epithelial transition (MET), confirmed by upregulation of the epithelial marker E-cadherin and downregulation of the mesenchymal marker vimentin. Importantly, this MET resulted in reduced colony formation (p<0.0042) and cell motility and dramatically reduced invasion (p<0.0001). Conversely, silencing of IGF-1R in epithelial MDA-MB-468 TN cells induced epithelial-to-mesenchymal transition (EMT), confirmed by downregulation of E-cadherin and upregulation of vimentin expression, with resultant increased colony formation (p<0.006), cell motility, and invasion (p<0.0001). Conclusion: Collectively, these results demonstrate a paradoxical effect of targeting IGF-1R in TN breast cancers of mesenchymal and epithelial origin. Targeting IGF-1R in TN breast cancers with a mesenchymal phenotype decreases invasion and metastatic potential. In contrast, targeting IGF-1R in TN breast cancers with an epithelial phenotype could have potentially detrimental effects. Our data suggest that IGF-1R inhibition should be explored as a therapeutic modality in TN breast cancers with a mesenchymal phenotype.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-06.
P1-02-07: Epithelial-Mesenchymal Transition Correlated with Serum Cytokine Profiling in Breast Cancer Patients.
Authors: Giordano A , Cohen EN , Anfossi S , Gao H , Lee B-N , Mego M , Sanda T , Valero V , Alvarez RH , Cristofanilli M , De Placido S , Hortobagyi GN , Woodward W , Ueno NT , Reuben JM
Published: June 2012

Background: Inflammation contributes to the increased invasiveness and poor prognosis in breast cancer (BC) patients. Specifically, the expression of the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα) and interleukin-1 (IL-1) have all been linked to increased invasiveness and poor prognosis. Interestingly, the increased invasiveness was associated with an increase in the acquisition of markers of epithelial-mesenchymal transition (EMT). Therefore, we determined whether the levels of circulating proinflammatory cytokines (IL-1, IL-6, TNFα) and antiinflammatory cytokines (IL10) were correlated with the induction of EMT transcription factors (TFs), Snail1, Zeb1, Twist1, in breast cancer patients.

Materials and Methods: From two laboratory-based ongoing studies at the MD Anderson Cancer Center, 41 BC patients were assessed for EMT-TFs in circulating CD45-ve cells (EMT-CTCs) and for serum proinflammatory cytokines before starting any treatment. 32 of 41 patients assessed for EMT had metastatic BC. EMT-CTCs were detected by qRT-PCR for the EMT-TFs Snail1, Zeb1 and Twist1 (Mego 2011; PMID 21387303) and serum cytokines were measured by Luminex bead array assay (MILLIPLEX™ MAP Human Cytokine/Chemokine Panel). Cytokine serum concentrations were compared with the median cytokine levels of healthy donors (HD). We examined the association of serum cytokines above the median HD levels and the presence of EMT-CTCs by non-parametric Mann-Whitney test with a statistical significance for p<.05.

Results: Of the 41 patients assessed for both serum cytokines and EMT-CTCs, 14 (34%) were positive for at least one EMT-TF, including 3 of 9 (33%) patients with no-metastatic BC and 11 of 32 (34%) patients with metastatic BC. We found that serum levels of IL1a, IL2, TGFα, and TNFβ in patients that were above the median levels of HD sera were higher in patients with EMT-CTCs in the blood (higher IL1a concentration in patients with over expression of Snail1, Zeb1, and Twist1; IL2 with Zeb1; TGFα with Snail1; TNFβ with Zeb1, and Twist1). Further, the higher ratio of proinflammatory/anti-inflammatory cytokines, was associated with the presence of at least one EMT-TF, e.g., IL8/IL10 (p=.005) and TNFα/IL10 (p=.037). Discussion: Patients with proinflammatory cytokine (IL1a, IL2, TGFα, and TNFβ) levels above the median levels of HD or who had a predominantly proinflammatory cytokine profile were more likely to have at least one EMT-TF in their blood. These data are consistent with the hypothesis that proinflammatory cytokines promote EMT, which may be involved in tumor aggressiveness and disease progression.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-07.
P1-02-08: Modulation of EMT by Targeting E-Cadherin Restores Radiation Sensitivity in Human Breast Cancer Cells.
Authors: Munshi A , Yuan Y , Liu J , Meyn RE
Published: June 2012

E-cadherin is the major adhesion protein associated with epithelial cells and loss of its expression is diagnostic of the epithelial to mesenchymal transition (EMT). Findings from our laboratory, suggest that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which typically are relatively radiosensitive. To definitively test this hypothesis we compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. We observed that many ER-a negative lines, were also E-cadherin negative, and the ER-a positive lines were positive for E-cadherin expression. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) and had high expression of E-cadherin, were also more sensitive to increasing doses of radiation. Treatment of MCF-7 cells with 100nM siRNA to E-cadherin for 72 hrs made the cells more resistant to radiation with the surviving fraction changing from 42.3% in control cells to 56.4% in siRNA treated cells. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 and Hs578t cells with a CDH1 -expression vector and isolated stable clones, which were then tested for radiosensitivity. Cells transfected with the backbone vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized both, MDA-MB-231 and Hs578t cells, compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. These observations suggest that ER-a signaling might regulate E-cadherin and implied that this regulation might influence EMT and radioresistance. Overall, our results suggest that the EMT status of breast cancer cells governs their response to radiation. A detailed investigation of the mechanism is ongoing.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-08.
P1-03-06: Fibroadenomatoid Changes Have a Higher Occurrence Rate in Middle-Aged Benign Breast Disease Patients with the Trend Retained in Cancer Patients.
Authors: Bekhash A , Hooke JA , Chen Y , Kovatich AJ , Kvecher L , Mural RJ , Shriver CD , Hu H
Published: June 2012

Background: Fibroadenoma (FA) is a common benign breast lesion known to have a high incidence rate in younger women. There are controversial reports whether FA elevates the risk of developing breast cancers. In clinical practice, FA may be surgically removed due to multiple reasons making it complicated to study its impact on the development of breast cancers that have a higher incidence rate in older women. Fibroadenomatoid change (FAC), also known as fibroadenomatous hyperplasia, is an uncommon lesion with histologic features similar to that of FA but lacking well-defined borders and usually discovered incidentally on breast biopsy specimens. FAC is not surgically targeted. The Walter Reed Army Medical Center, through the Clinical Breast Care Project, has enrolled over 2000 subjects undergoing a biopsy; all the pathology was reviewed by a single pathologist. These subjects provide an opportunity to study the age-dependent pattern of FAC in different patient populations.

Methods: Subjects were enrolled following IRB-approved protocols with data collected through two comprehensive questionnaires, a Core Questionnaire and a Pathology Checklist. A total of 1964 female subjects were identified for this study, including 1135 benign/atypical, 192 in situ, and 637 invasive cancer patients. Patients were divided into three age groups: <=45 years, 46–65 years, and >=66 years. Chi-Square test in the SAS was used for statistical analysis.

Results: As shown in the table, FA occurrence rate decreases significantly with increasing age in benign disease patients. FAC, on the other hand, shows a significantly higher occurrence rate in middle-aged patients with benign findings, and this trend is retained in the invasive or in situ cancer populations. FAC rate is also significantly higher in patients with cancer (invasive, or invasive and in situ combined) compared to benign patients in each age group with p-values ranging from 0.0001 to 0.019 (not shown).

Discussion: Our preliminary results suggest that FAC occurs more often in middle-aged patients. It's significantly lower occurrence in patients with benign findings may be partially explained by the fact that breast cancer patients undergo more extensive surgeries, thus providing more breast tissue for pathologic evaluation. Otherwise, the increased FAC rate may suggest its role as a risk factor for cancer development. Since FAC may be considered a miniature FA that is not surgically targeted, it may be used as a window for the study of FA on its impact in cancer development. Further study needs to be performed to explain why FA and FAC have different age-dependent patterns and whether FA or FAC is a risk factor for breast cancer development.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-06.
P1-04-05: T-DM1 and Pertuzumab as New Tools for HER2 Specific Antibody-Therapy Against Breast Cancer Stem Cells in HER2−Positive Mammary Carcinoma.
Authors: Honig A , Diessner J , Dietl J , Wischhusen J
Published: June 2012

Trastuzumab (Herceptin®) has significantly improved the overall survival of patients with HER2−positive breast cancer. The efficacy of this treatment depends significantly on antibody-dependent cell mediated cytotoxicity (ADCC). However, while trastuzumab can direct NK cells towards tumor cells and thereby promote tumor cell lysis, spheroid-forming tumor-initiating cells are largely spared. Consequently, populations of surviving cells showed greatly enhanced clonogenicity in vitro and tumorigenicity in vivo. Low HER2 levels and reduced expression of activating NK cell ligands provide likely explanations for the escape of these cells from HER2−directed NK cell-dependent therapies. Moreover, recent data show that unproductive interactions between immune cells and cancer cells can induce a process called “epithelial-to-mesenchymal transition” (EMT) which enables previously differentiated cancer cells to acquire stem cell-like properties as evidenced by a CD44highCD24low breast cancer stem cell signature and aldehyde dehydrogenase positivity. We thus investigated whether new HER2−specific antibodies could enhance NK-cell mediated target cell lysis or inhibit the induction of tumor stem cells.

The antibody pertuzumab also binds to the HER2−receptor. It inhibits homo- and heterodimerization of the HER2 receptor with other member of the HER family, in particular the dimerization of HER2/HER3 receptors. We were able to show that the combination of trastuzumab and pertuzumab leads to increased antibody-binding on breast cancer cells and breast cancer stem cells, resulting in enhanced ADCC. This correlates with the clinically observed synergy between trastuzumab and pertuzumab as observed for example in the Neosphere trial, a neoadjuvant study for HER2−positive breast cancer. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate which combines trastuzumab with the tubulin inhibitor maytansine. Interestingly, treatment with T-DM1 largely prevented the NK cell-dependent induction of the EMT markers Twist and Snail and the characteristic downregulation of E-cadherin. Thus, T-DM1 apparently interferes with EMT and may hence prevent the re-induction of the stem cell phenotype. In line with the presumed effect against cancer stem cells, T-DM1 significantly attenuated the clonogenic potential of FACS-sorted trastuzumab-unresponsive cancer stem cells. Accordingly, our data show that these new therapeutics may be more effective against cancer stem cells and thereby promise a more sustained treatment of HER2−positive breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-05.
P1-05-03: Relationship between Polycomb Repressive Complex EZH2/CBX7, Large Non-Coding RNA ANRIL and Stem Cells Biomarkers in Triple Negative Breast Carcinomas: Important Role in Carcinogenesis through an Epigenetic Silencing Process and New Clues for Targeted Therapies.
Authors: Meseure D , Vacher S , Trassard M , Drak-Alsibai K , Le Scodan R , Le Ray C , Regnier C , Lidereau R , Bieche I
Published: June 2012

Background: Epigenetic deregulation and carcinogenesis are intimately connected and gene silencing is a major consequence of epigenetic modifications. Polycomb group proteins (PcG) play important roles by inhibiting chromatin remodeling and transcription, silencing tumor suppressor genes, regulating stem cells and interconnecting with Wnt/beta-catenin, TGF-beta and Sonic-Hedgehog pathways. EZH2 is a transcriptional repressor and acts mainly as an oncogenic silencer gene involved in cell cycle regulation. Large non coding RNA are major regulators of oncogenic and oncosuppressive pathways at epigenetic, transcriptional and post-transcriptional levels. Recent studies have showed that (i) EZH2 overexpression is associated with cancer progression and aquisition of stem cell properties and (ii) disruption of ANRIL/CBX7 interactions repress the INK4b/ARF/INK4a locus. The aim of the study was to analyze PRC complexes, large non-coding RNA ANRIL and downstream signaling targets in triple negative breast carcinomas (TNC).

Design: By using real time quantitative RT-PCR in a series of 432 invasive breast ductal carcinomas (IDC), we quantified gene expression levels of the large non coding RNA ANRIL, PcG (EZH2, CBX7, SUZ12), stem cells markers (ALDH1, CD133, ASCL1) and oncosuppressor genes (p15, p16 and p19). Immunohistochemistry (IHC) was performed using EZH2, CBX7, ALDH1 and b-catenin Abs. We compared mRNA expression levels and IHC intensity score in TNC with non TNC and normal breast parenchyma.

Results: EZH2 overexpression was respectively found by RT-PCR and IHC in the TNC subgroup of IDC. We observed variable mRNA expression levels of SUZ12, CBX7, ANRIL, ALDH1, CD133, ASCL1, p15, p16, and p19. IHC showed EZH2 and CBX7 nuclear staining. Mutual positive correlations were found between EZH2, CBX7, ALDH1, ASCL1 and CCND1 (p<10-5).

Conclusion: Polycomb EZH2/CBX7 and the large non coding RNA ANRIL are epigenetic transcriptional repressors overexpressed in the TNC subgroup of IDC. Their implication in the process of gene silencing (p16, p15, p19), activation of oncogenic pathways (Wnt, TGFb) and tumor stem cells self-renewal may explain several hallmarks of TNC and open new avenues to target this aggressive subgroup.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-05-03.
P1-05-04: Distinct Patterns of Promoter CpG Island Methylation of Breast Cancer Subtype Are Associated with Different Stem Cell Phenotype.
Authors: Park SY , Kwon HJ , Choi Y , Lee HE , Kim S-W , Kim JH , Kim IA , Jung N , Cho N-Y , Kang GH
Published: June 2012

Background: Although DNA methylation profiles in breast cancer have been connected to breast cancer molecular subtype, there have been no studies of the association of DNA methylation with stem cell phenotype. This study was designed to evaluate promoter CpG islands methylation of 15 genes with regard to breast cancer subtype and to investigate whether the patterns of CpG island methylation in each subtype are associated with cancer stem cell phenotype represented by CD44+/CD24- or ALDH1 expression.

Methods: We performed MethyLight analysis for the methylation status of 15 promoter CpG island loci involved in breast cancer progression (APC, DLEC1, GRIN2B, GSTP1, HOXA1, HOXA10, IGF2, MT1G, RARB, RASSF1A, RUNX3, SCGB3A1, SFRP1, SFRP4, and TMEFF2) and determined cancer stem cell phenotype by CD44/CD24 and ALDH1 immunohistochmeistry in 36 luminal A, 33 luminal B, 30 luminal-HER2, 40 HER2 enriched, and 40 basal-like subtypes of breast cancer.

Results: The number of CpG island loci methylated was significantly different among subtypes and it was highest in luminal-HER2 subtype and lowest in basal-like subtype. Methylation frequencies and levels in 12 out of the 15 genes were significantly different among all subtypes and basal-like subtype showed significantly lower methylation frequencies and levels in nine genes, compared to luminal A, luminal B, HER2 enriched, and luminal-HER2 subtypes. CD44+/CD24- or ALDH1+ putative stem cell populations were most enriched in basal-like subtype. The methylation of promoter CpG islands was significantly lower in CD44+/CD24-cell (+) tumors, compared to CD44+/CD24-cell (−) tumors, even within the basal-like subtype. ALDH1 (+) tumors also had significantly lower methylation, compared to ALDH1 (−) tumors.

Conclusions: Our findings showed that promoter CpG island methylation was significantly different according to breast cancer subtype and stem cell phenotype of tumor, suggesting that breast cancers have different methylation patterns according to molecular subtypes and it is associated with stem cell phenotypes of the tumor.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-05-04.
P1-05-06: Hypermethilation 14-3-3- Sigma Promoter as a Biomarker to Screening for Metastasis and Potencial Prognostic Factor in Breast Cancer Patients.
Authors: Martinez-Galan J , Torres-Torres B , González-Vicente A , Ruiz-Vozmediano J , Delgado-Pérez JR
Published: June 2012

Background: 14-3-3sigma is an epithelial marker whose expression is induced by DNA damage through a p53-dependent pathway. 14-3-3sigma functions sequesters cyclin B1-CDC2 complexes outside the nucleus and thereby contributes to a G2 arrest. Epigenetic silencing by CpG methylation, p53 inactivation, and proteasome-dependent proteolysis leads to loss of 14-3-3sigma and is often observed in precancerous lesions and likely to be causally linked to the onset of cancer

Objetive: To correlation methylation levels of promoter 14-3-3 sigma with association prognostic factors in breast cancer and their correlation with phenotype luminal.

Material and Methods: This is a prospective study we quantified methylation levels of promoter 14-3-3 sigma gene in 107 women with breast cancer and 108 control subjects by Real Time QMS-PCR SYBR green (methylation-specific PCR) 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%). Significant differences between breast cancer patients (pts) and healthy controls in relative serum levels of methylated gene promoters 14-3-3s (p=0.0047) were detected. Presence of methylated 14-3-3-s in serum of breast cancer patients was associated with T3-4 stage (OMS) (p<0.05) and nodal positive status (p< 0.05). With a median follow up 6 years we saw more probability of developing distance metastasis in patients with methylation 14-3-3 sigma (p>0.05).

Conclusions: Hypermethylation of the 14-3-3 sigma promoter is an early and frequent event in breast neoplastic transformation, leading to the suggestion that silencing of 14-3-3 sigma may be an important event in tumor progression and particularly in breast carcinogenesis. This study identifies the presence of variations in global levels of methylation promoters genes in patients breast cancer with different phenotype classes and shows that these differences have clinical significance. Perhaps in the detection of CpG methylation of 14-3-3sigma may be used for diagnostic and prognostic purposes.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-05-06.
P1-06-10: Lobular Breast Cancer and NAC: Combined Results from the NKI and I-SPY 1 Trial.
Authors: Mukhtar RA , Lips E , Wesseling J , Livasy C , Yau C , Berry D , van't Veer L , Carey LA , Esserman LJ , Rodenhuis S , Hwang ES
Published: June 2012

Background

The benefits of neoadjuvant chemotherapy (NAC) in breast cancer are twofold: allowing breast conservation surgery, and assessment of response. Having pathologic complete response (pCR) predicts improved outcomes. Many have suggested that invasive lobular cancers (ILC) do not respond to NAC as well as invasive ductal cancers (IDC), and recommend against offering NAC in such cases. We previously investigated differences in the response to NAC in ILC and IDC in the I-SPY 1 Trial, and now present findings from a joint analysis with the Netherlands Cancer Institute (NKI).

Methods: We combined datasets comprising 676 patients (221 from I-SPY, 455 from NKI) enrolled in NAC trials. Eligible patients had palpable tumors ≥ 3 cm, and underwent serial biopsies, microarray analysis (Agilent 44K for I-SPY and Illumina 6v3 for NKI), and MRI imaging. ILC versus IDC histology was assigned by pathologic appearance, and ILCs were tested for e-cadherin expression and centrally reviewed by a breast pathologist at each site. We performed multivariate logistic regression analyses to assess differences in pCR. We compared intrinsic subtypes, prognostic gene signature expression, and MRI phenotypes.

Results: There were 75 ILC and 601 IDC cases. ILCs had lower risk features and were more likely hormone receptor + (Table 1) but there was substantial heterogeneity and many ILC had high risk features. There were fewer T2 (33% versus 48%) but more T3 tumors (59% versus 37%) among the ILCs. Nearly 10% of ILC cases had the basal intrinsic molecular subtype; 22% expressed the activated wound healing signature, and 56% had a high risk 70-gene prognostic signature. While pCR rate was lower in ILCs, adjustment for low risk features showed lobular histology was not an independent predictor of pCR. ILC was less likely to present as a solid mass. 85% of ILC were e-cadherin negative.

Conclusions: This combined analysis suggests that the decreased response to NAC in ILC is due to the association between ILC and low risk features. ILC in a clinically high risk setting has a higher proportion of high risk biology in spite of lobular histology. Lobular histology alone should not be used to exclude patients from NAC trials or treatment.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-10.
P1-06-12: Circulating Tumor Cells (CTC) Monitoring during Phase II Study with Lapatinib (L) and Capecitabine (C) in Patients with Brain Metastases from HER2−Positive (+) Metastatic Breast Cancer (MBC) before Whole Brain Radiotherapy (WBR): LANDSCAPE Study.
Authors: Pierga J-Y , Cropet C , Tresca P , Dalenc F , Romieu G , Campone M , Mahier Aït-Oukhatar C , Le Rhun E , Gonçalves A , Leheurteur M , Domont J , Gutierrez M , Curé H , Ferrero J-M , Labbe-Devilliers C , Bidard F-C , Bachelot T
Published: June 2012

Background: Decrease of CTC level during treatment in MBC has been reported as an independent prognostic and predictive factor of patients’ outcome. Monitoring CTC in addition to clinical response criteria is currently evaluated in early clinical trials in various cancer types. We sought to evaluate the clinical interest of peripheral blood CTC for patients included in the LANDSCAPE study which assessed the efficacy of upfront systemic treatment with L+C for newly diagnosed brain metastasis.

Methods: This analysis is a preplanned secondary endpoint of the LANDSCAPE study. Eligible pts had HER2+ MBC with BM not previously treated with WBR, C or L. Pts received L1250 mg/day and C2000 mg/m2/day, days 1–14, every 21 days. The primary endpoint was a centrally assessed CNS objective response (CNS-OR) defined as a ≥50% volumetric reduction of CNS lesions in the absence of increasing steroid use, progressive neurologic symptoms or progressive extra-CNS disease. CTC were detected in 7.5 ml of blood using the CellSearchSystem™, combining EpCAM immunomagnetic selection (IMS) followed by anti-cytokeratin (A45B/B3) fluorescently staining for CTC at baseline and at day (D) 21, before cycle 2.

Results: From 04/2009 to 08/2010, 45 pts were enrolled, 41 were evaluable for CTC at baseline and 38 at D21. Median age was 56 (range 35 to 79). PS was >1 only in 2 pts. At baseline, 20/41 (48.8%) pts had ≥ 1CTC and 9 (22%) ≥ 5CTC (range 1–301, median 3). CTC were detected in pts with (18/37) or without disease outside SNC (2/6) (p=0.63). At a median follow-up of 10 months (range 2.9−16.5), median TTP was 6.0 months [95% CI 4.9; 7.4] vs. 4.3 [2.8; 5.9] months for pts without and with CTC at baseline respectively (p=0.14). After 21 days of treatment, a disappearance of CTC was observed in 11pts (31%). At D21, only 7 (18.4%) pts had ≥ 1CTC and 3 (8%) ≥ 5 CTC (p=0.006, D21 vs. baseline). In 43 evaluable pts, CNS-OR rate was 67% (95%CI 51–81), with a median time from inclusion to response of 1.8 month. Absence of CTC was not correlated with CNS-OR rate at baseline (17/21 (81%) vs. 11/19 (58%), NS). Strikingly, remaining positivity for CTC at D 21 (≥ 1CTC) was correlated with a poor response rate in CNS: 2/6 (33.3%) vs. 25/31 (80.6%) in pts with 0 CTC, p=0.03.

Conclusions: Early decrease (at D 21) in CTC level is correlated with a high response rate in newly diagnosed BM to L + C and underlines the predictive value of this blood marker in MBC pts even for brain metastasis. Longer follow-up is needed to assess its prognostic value under antiHER2 targeted therapy.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-12.
P1-06-15: A Genomic Predictor Developed from Breast Cancer Cell Lines Predicts Both Disease-Free Survival and Overall Survival in Breast Cancer Patients Treated with Doxorubicin and Cyclophosphamide: A Collaborative Project of the NSABP and Precision Therapeutics.
Authors: Shen K , Tang G , Kim C , Pogue-Geile K , Anderson SJ , Constantino JP , Bear HD , Song N , Tian C , Gabrin MJ , Zhang Y , Geyer CE , Wolmark N
Published: June 2012

Background: A cell line-derived multigene predictor of tumor response to doxorubicin + cyclophosphamide (MGP-AC) has been shown to predict the pathological complete response (pCR) in breast cancer patients from the National Surgical Adjuvant Breast and Bowel Project (NSABP) Protocol B-27. However, a cell line-derived MPG for doxorubicin + cyclophosphamide + docetaxel (MGP-ACT) was not predictive in patients from B-27. The purpose of this study was to further assess the performance of these predictors on disease-free survival (DFS) and overall survival (OS) in the same patient populations.

Methods: NSABP B-27 was a 3-arm trial of 2411 early-stage breast cancer patients randomized to receive 4 cycles of preoperative doxorubicin+cyclophosphamide (AC) or 4 cycles of AC followed by 4 cycles of docetaxel either pre-op (AC+T) or post-op (AC→T). MGPs for AC and ACT were developed based on the in vitro assay and microarray genomic profiles of 40 breast cancer cell lines. A higher MGP score indicates lower chemoresponse sensitivity.

Results: 322 patients with available microarray data were included for this analysis (103 treated with AC, 102 with AC+T, and 117 with AC→T). For patients treated with AC, a higher MGP-AC score was significantly associated with increased risk of disease progression (standardized hazard ratio [HR] [SD set to 1]=1.48, 95% confidence interval [CI]=1.02−2.15, p=0.043) or death (standardized HR=1.66, 95% CI=1.06−2.62, p=0.028) after adjusting for clinical covariates (ER status, clinical tumor size, lymph node status, and age). The addition of MGP-AC to the clinical model improves the accuracy in predicting five-year DFS: the area under the ROC curve improved from 63% to 72%. For patients treated with AC+T or AC→T, MGP-ACT was not predictive of either DFS (standardized HR=1.03, 95% CI=0.78−1.37, p=0.818) or OS (standardized HR=1.05, 95% CI=0.73−1.51, p=0.8).

Conclusions: A cell line-derived MGP for AC that was predictive of pCR was also predictive of DFS and OS in breast cancer patients treated with neoadjuvant AC. The MGP for ACT, which was not predictive for pCR, was not predictive of either DFS or OS in patients who received docetaxel after AC.

The B-27 study was funded by NCI PHS grants U10-CA-37377, U10-CA-69974, U10-CA-12027, U10-CA-69651, and U24-CA-114732, and received additional support from sanofi-aventis.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-15.
P1-07-04: Gene Expression Module Biomarkers To Stratify Multiple Clinical and Therapeutic Endpoints for Universal Breast Cancer Companion Diagnostic.
Authors: Rhodes D , Tomlins S , Williams P , Sadis S , Wyngaard P , Oades K , Chattopadhyay S , Wang Y , Monforte J , Lee B-I
Published: June 2012

Gene expression patterns are increasingly capable of stratifying patients based on prognosis and response to therapy. Given the limited availability of sample tissue, however, it is not feasible to run many tests, suggesting the need for a universal companion diagnostic assay that is informative with respect to multiple clinical and therapeutic endpoints. Key challenges are identification of appropriate gene expression biomarkers, translation of biomarkers to clinical assays, and development of reliable gene expression profiling of formalin-fixed clinical specimens. Here, we describe a meta-analysis approach that identifies novel biomarker modules that results in multiple clinical and therapeutic read-outs.

A co-expression meta-analysis of 5,339 breast tumors from 56 microarray datasets identified highly co-expressed sets of genes (modules) across multiple datasets. These module based biomarkers were tested for their ability to associate with prognostic and predictive targets in published datasets. In addition, each module was reduced from 10 - 1,000 genes to the top performing 2–3 genes based on the degree of co-expression across the meta-analysis and validation by quantitative PCR in an independent panel of FFPE tumor samples. This study demonstrates that a single 96 gene qPCR test utilizing multiple module biomarkers is not only capable of stratifying patients by standard histopathological parameters (ER, PR and Her2), but also stratifies by other diverse elements of the disease (cell lineage, dysregulated core biological functions, factors of cell growth, underlying genomic aberrations and the tumor microenvironment). Taken together, these biological variables represent the major biological diversity present within the breast cancer population. A series of retrospective analyses demonstrated that different single module and combinations of modules were capable of predicting a variety of clinical endpoints, including 5-year survival, neoadjuvant chemotherapy response in ER- patients and targeted therapy response in model systems.The molecular heterogeneity of breast cancer can be summarized by discrete gene expression modules that individually represent distinct biological pathways, and that collectively can be represented by as few as 96 genes. These breast cancer modules, together with outlier genes, allow for summation of the entire transcriptional program and provide a universal assay with broad application to companion diagnostics development.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-04.
P1-07-05: HER2 Status Resolution in FISH and IHC “Double Equivocal” Breast Carcinomas by Quantitative Real-Time PCR.
Authors: Portier BP , Wang Z , Downs-Kelly E , Budd GT , Lanigan C , Tubbs RR
Published: June 2012

Background: Clinical testing for HER2 amplification/over-expression is performed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) as outlined by the ASCO/CAP guidelines. Although these guidelines standardize testing and reporting, in a subset of patients, HER2 is equivocal by both IHC and FISH (“Double Equivocal”). These double equivocal patients represent a clinically problematic sub-group that currently lack standardized management guidelines. In this study, we utilize Quantitative Real-Time PCR (Q-RT-PCR) to resolve HER2 status in invasive breast cancer cases that could not be resolved via IHC and FISH testing.

Material and Methods: FISH for HER2 was performed on 2259 invasive breast carcinomas from 1/2008 to 12/2010. In accordance with ASCO/CAP, all equivocal HER2 FISH cases were reflex tested by IHC. In double equivocal cases, RNA extraction was performed following macro-dissection using High Pure RNA Paraffin Kit (Roche Applied Biosciences, Indianapolis, IN). Q-RT-PCR was carried out using TaqMan® RNA-to-CT™ 1-Step Kit with primers and probes for HER2, B2M, and GAPDH (Applied Biosystems, Foster City, CA). Q-RT-PCR results were expressed as the relative quantification of HER2 vs. two control genes, all normalized against calibrator RNA from the MCF7 cell line. Cut off for Q-RT-PCR HER2 overexpression was set using ROC curve analysis (MedCalc, Belgium).

Results: In our cohort of 2259 patients, 124 (5.5%) had an equivocal HER2 result by primary FISH testing. Reflex HER2 testing by IHC was unable to resolve the HER2 status in 35 (1.5%) patients. Detection of HER2 overexpression by Q-RT-PCR was validated using 50 FISH confirmed amplified and 50 non-amplified cases. Q-RT-PCR performed on these 2 control populations generated two non-overlapping populations and ROC curve analysis using a cut off value of 7.0 showed 100% sensitivity and specificity in detection of HER2 overexpression. Application of Q-RT-PCR in the double equivocal sub-group resulted in resolution of HER2 status in all cases, 8 HER2 positive (test value ranging from 7.12 - 15.37) and 14 HER2 negative (test value ranging from 1.05 - 6.92).

Conclusion: Application of Q-RT-PCR for HER2 represents a viable approach to resolve HER2 status in cases that fail classification by both FISH and IHC. Q-RT-PCR combines the precision and high sensitivity of real-time PCR with the morphological specificity of histological evaluation and ultimately allows definitive HER2 classification at the time of initial diagnosis. This knowledge of HER2 status at the time of diagnosis allows for comprehensive neoadjuvant treatment although, additional studies correlating response to anti-HER2 therapy and HER2 status by Q-RT-PCR are warranted.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-05.