bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1-05-03: A requirement for neural precursor cell-expressed developmentally downregulated gene 9 during the initiation of mammary tumorigenesis in MMTV-neu mice
Authors: Serzhanova VA , Little JL , Izumchenko E , Seo S , Kurokawa M , Egleston B , Klein-Szanto AA , Golemis EA
Published: August 2014

The Neural precursor cell-Expressed Developmentally Downregulated gene 9 (NEDD9; also HEF1, CAS-L) scaffolding protein regulates many signaling pathways associated with mitosis, survival, migration, and ciliary integrity. Within the last few years, elevated NEDD9 expression has been implicated in progression and metastasis of several types of cancer, including those of the lung, skin, and brain. We have investigated the consequences of introducing a Nedd9 genotype into the MMTV-neu mouse mammary tumor model, which in many respects recapitulates features of human HER2+ breast cancer. 80% of wild-type MMTV-Neu; Nedd9+/+ animals developed tumors with an average latency of 339 days, but only 18% of MMTV-Neu;Nedd9−/− animals developed tumors with an average latency of 416 days. This highly significant difference indicates that the Nedd9−/− genotype significantly prevents neu-dependent mammary tumor formation.

HER2-positive human breast tumors and MMTV-neu-induced tumors originate from mammary luminal epithelial progenitor cells. We evaluated mammary progenitor cell populations from non-tumor bearing MMTV-neu;Nedd9−/− versus MMTV-neu;Nedd9+/+ 4-month old mice (2 months prior to appearance of tumors). Flow cytometry analysis indicated a significantly reduced CD24high;CD49flow luminal progenitor subpopulation in MMTV-neu;Nedd9−/− and Nedd9−/− mammary glands, that could be contributed to the tumorigenesis resistance in the MMTV-neu;Nedd9−/− model. The MMTV-neu;Nedd9−/−genotype negatively affected the Matrigel mammosphere colony-forming potential of luminal progenitor cells compared to MMTV-neu; Nedd9+/+cells, causing formation of fewer colonies with aberrant size and morphology. Quantification of mitotic division planes of MMTV-neu;Nedd9−/− mammospheres revealed no contribution to the observed defects. However, MMTV-neu;Nedd9−/− mammospheres had defective expression of signaling proteins governing cell attachment, with reduced levels of FAK and more cytoplasmic localization of SRC.

The results reported above, together with other data, support three main conclusions. First, they revealed a very substantial requirement for Nedd9 during early stages of HER2/neu-dependent tumor formation. Second, these results are the first to demonstrate a role for Nedd9 in supporting the abundance and colony-forming potential of mammary luminal progenitor cells. Third, they indicate that the defects in mammosphere growth likely involve perturbation of crucial cellular attachment signaling pathways involving FAK and SRC. In sum, these data provide a strong justification for future analysis of NEDD9 in the defective signaling of transformed mammary epithelial progenitor cell populations that initiate human breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-03.
Abstract P1-05-20: Fluorescent Hyaluronan Probes Distinguish Heterogeneous Breast Cancer Cell Subsets and Predict their Invasive Behavior
Authors: Veiseh M , Kwon DH , Borowsky AD , Toelg C , Leong H , Lewis J , Turley EA , Bissell MJ
Published: August 2014

Background: Tumor heterogeneity is a determining factor in diagnosis and therapy of breast cancer (Bca). We investigated Bca cellular heterogeneity by a functional fluorescent hyaluronan (HA) probe, and monitored its binding characteristics in relation to the degree of Bca aggression. HA is a polysaccharide ligand for CD44 and RHAMM receptors, which are linked to cell plasticity and predict poor clinical outcome in Bca.

Methods: Fluorescent HA probes, multiplexed profiling, and cell sorting strategies were developed and used to detect/capture tumor cell subpopulations with differences in HA binding (HA−/low vs. HAhigh) and HA receptors surface display. Whereas we concentrated on the most metastatic cell line, MDA-MB-231, we also monitored the presence of subpopulations in other Bca lines and showed heterogeneity in all.

Results: The HA probe binding was highly heterogeneous, and the highest binding level was detected in highly invasive triple-negative basal subtypes such as MDA-MB-231. HA probes bound to the cell surface but also accumulated in cytoplasm and nucleus. Binding levels were dramatically reduced upon ‘reversion’ of highly malignant cells to a non-malignant phenotype in three-dimensional cultures, suggesting that the level of cellular binding to fluorescent HA probe provides a measure of malignant behavior. Comparison between HAhigh and HA−/low subpopulations revealed surprising differences in morphology, proliferation and invasion in culture, which were retained in two in vivo models. HAhigh subpopulations exhibited higher levels of invasion but surprisingly lower levels of proliferation compared to either unsorted parental cells or the HA−/low subpopulation.

Conclusions: Querying HA binding in Bca lines reveals an unexpected heterogeneity with respect to tumor phenotype in vivo, morphology in 3D, invasion and proliferation. These results may aid in diagnosis and therapy of invasive Bca subpopulations and early sorting of cancer patients needing intensive chemotherapy.

Keywords: Heterogeneity, 3D cultures, Hyaluronan, CD44, RHAMM/HMMR, Tumor phenotype, Breast cancer, Invasion.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-20.
Abstract P1-09-03: Chemoprevention in patients with newly diagnosed breast cancers
Authors: Refinetti AP , Chun J , Schnabel F , Guth A , Axelrod D
Published: August 2014

Background: Chemoprevention (including tamoxifen, raloxifene, and exemestane) is a strategy to reduce breast cancer incidence in high risk women. Studies have shown at least a 50% decrease in the incidence of breast cancer in users of these drugs. Despite this benefit, the majority of high risk, unaffected women who are offered chemoprevention decline the therapy. However, there is a growing population of women who have used these agents for primary prevention, and a larger population of survivors who have used these drugs as part of their systemic treatment. The purpose of this study was to identify a cohort of women with newly diagnosed breast cancers who had utilized chemoprevention and describe their patterns of disease.

Methods: The Breast Cancer Database of NYU Langone Medical Center was queried for patients who used chemopreventive drugs and developed breast cancer between 1/2010-1/2012. Patients were divided into primary and secondary chemoprevention groups (no previous history of breast cancer and previous history of breast cancer, respectively). Descriptive statistics were utilized.

Results: In the study period, 24 (2%) of 996 patients had used a chemopreventive agent. For 16 of the 24 (67%), the drug was part of systemic therapy for prior breast cancer, with a median of 12 years from the initial diagnosis to the diagnosis of a second breast cancer. The primary chemoprevention group included women with risk based on family history and atypical hyperplasias. The majority of patients were diagnosed with early stage disease (88% DCIS and stage I). This likely reflects their screening behaviors. In both groups, the majority of cancers were ductal in origin. Five of the 8 patients in the primary chemoprevention group were on treatment at the time of their cancer diagnosis, while 63% of patients in the secondary group were prior users. In the secondary group, the majority of cases were contralateral second primary breast cancers, with 31% ipsilateral recurrences. Interestingly, the majority of cancers in both groups were ER/PR positive.

Conclusions: Our cohort of women who used chemoprevention drugs were overwhelmingly diagnosed with early stage breast cancer, likely reflecting their commitment to screening. The majority of cancers were ER/PR positive. In this group, the choice of cancer treatment will need to be modified in light of prior hormonal treatment. Many of the patients in the secondary group were past users of prevention agents and further work is needed to clarify the duration of benefit of these drugs. In a similar vein, we look forward to research efforts to define the optimal age to initiate primary chemoprevention in high risk women.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-09-03.
Abstract P1-09-06: Biological Effects of Green Tea Capsule Supplementation in Pre-surgery Breast Cancer Patients
Authors: Yu S , Spicer D , Hawes D , Wu A
Published: August 2014

Green tea is rich in antioxidant polyphenols consisting primarily of epicatechins, epigallocatechin, epicatechin gallate, and epigallocatechin gallate (EGCG). There are suggestive evidence from observational epidemiologic studies that regular green tea consumers may have lower risk of breast cancer development and mortality. Inhibition of cell proliferation and angiogenesis and enhancement of apoptosis are some of the mechanisms that have been proposed to explain the anti-cancer actions of the bioactive extracts of green tea. We conducted a randomized pre-surgical pilot study of green tea capsules in women diagnosed with breast cancer at the Los Angeles County-University Southern California Medical Center. Women in both the green tea and control arms were non-green tea drinkers at baseline (i.e. less than 1 cup of green tea per week at the time of breast cancer diagnosis). Women (n = 17) who participated in the green tea arm took 3 green tea capsules daily (Pro Health Green Tea Mega EGCG, 3 capsules contained ∼ 870 mg EGCG; equivalent to ∼ 8 cups of green tea) between their breast cancer diagnosis until their scheduled surgery date. The average duration of green tea intake was 35 days. Women in the control arm did not drink green tea between diagnosis and surgery date. Participants in the green tea and control groups donated blood and urine specimens at enrollment and before surgery which were tested for urinary tea catechin levels as a measure of compliance. Baseline urinary tea catechin concentrations were low in both green tea and control arms. Tea catechin concentrations remained low for women in the control arm but increased significantly (2–10 folds for different catechins) for women in the green tea arm. The differences in pre-surgery urinary green tea levels were significantly different between these groups of participants We conducted standard immunohistochemical analysis of the formalin-fixed paraffin-embedded respective markers of cell proliferation (Ki67), apoptosis (BAX), and angiogenesis (CD34). Separate analysis was performed on benign and malignant breast cancer tissue. The changes in these three biomarkers pre- and post-treatment in the two groups will be presented.

Supported by the WHH Foundation.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-09-06.
Abstract P1-12-02: Results of a phase 2, multicenter, single-arm study of eribulin mesylate as first-line therapy for locally recurrent or metastatic HER2-negative breast cancer
Authors: Vahdat L , Schwartzberg L , Glück S , Rege J , Liao J , Cox D , O'Shaughnessy J
Published: August 2014

Background: Eribulin mesylate is a novel nontaxane microtubule dynamics inhibitor that is approved in patients (pts) with metastatic breast cancer (MBC) who have previously received at least two chemotherapeutic regimens for MBC. We are reporting a preliminary planned analysis (first 6 cycles of treatment) of a phase 2 study that evaluates efficacy and safety of eribulin as first-line therapy for HER2-negative MBC.

Methods: Pts with measureable HER2-negative locally recurrent or MBC with at least 12 months since prior neoadjuvant or adjuvant chemotherapy received eribulin mesylate at 1.4 mg/m2 IV on days 1 and 8 of each 3-week cycle. Endpoints include objective response rate (ORR) (primary), safety, progression free survival (PFS), time to response (TTR), and duration of response (DOR). Tumor assessments were evaluated every 6 weeks for the first 6 cycles and every 6–12 weeks thereafter per RECIST 1.1.

Results: 54 of 56 enrolled pts had at least 1 post-baseline assessment. The median number of cycles delivered was 7 (range 1,21). Pt characteristics: median age, 56 yrs (range 31–85); ECOG of 0, 32 (57%); 29% had de novo stage IV; 38/56 (68%) had prior neo/adjuvant (45% had anthracycline and 45% taxane, the majority of which were given together). Seventy percent have visceral disease (45% liver, 38% lung); 41 (73%) have estrogen receptor-positive (ER) disease and 12 (21%) have triple negative (TN) (ER−/PR−/HER2−) disease. Objective response data are found in Table 1.

The most common treatment-related AEs are reported in Table 2.

Treatment-related serious AEs occurred in 5 (9%) pts, neutropenia (4%), and febrile neutropenia (5%). Five pts stopped study therapy due to adverse events. Fourteen pts remain on study treatment.

Conclusions: The preliminary results of this first-line study suggest that eribulin has antitumor activity in ER+/HER2− and TN MBC with an acceptable safety profile. Further exploration of this treatment as part of neo/adjuvant therapy is warranted.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-12-02.
Abstract P1-13-02: Analysis of the NNBC 3-Europe trial: Addition of docetaxel to anthracycline containing adjuvant chemotherapy in high risk node-negative breast cancer patients.
Authors: Thomssen Christoph , Kantelhardt Eva-Johanna , Meisner Christoph , Vetter Martina , Schmidt Marcus , Martin Pierre-Marie , Veyret Corinne , Augustin Doris , Hanf Volker , Paepke Daniela , Sweep Fred CGJ , Schmitt Manfred , von Minchwitz Gunter , Jaenicke Fritz , Harbeck Nadia
Published: August 2014

Withdrawn by Author

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-13-02.
Abstract P1-14-03: Overall survival results of a multicenter randomized phase II study in locally advanced breast cancer patients treated with or without neoadjuvant Trastuzumab for HER2 positive tumor (Remagus 02 trial)
Authors: Giacchetti S , Pierga J-Y , Asselain B , Delaloge S , Brain E , Espié M , Mathieu M-C , Bertheau p , de Cremoux P , Tembo O , Marty M
Published: August 2014

Background: Trastuzumab is indicated in neoadjuvant setting in locally advanced HER2 positive breast cancer patients (Gianni L. Lancet 2010). There is no data on the impact of the use of neoadjuvant Trastuzumab (T) compared to adjuvant T on survival.

Patients and methods: From May 2004 to October 2007, 341 stage II-III breast cancer patients were included in a phase II randomized trial and received 4 cycles (c) of epirubicin (75 mg/m2 d1)–cyclophosphamide (750 mg/m2 d1) q 3 w followed by 4 (c) of docetaxel (100 mg/m2 d1) q 3 w. Pts with HER2+++ tumor (120 pts) were randomized to receive or not neoadjuvant T combined with docetaxel. From September 2005, all pts with HER2+cancer received adjuvant T for a total of 18 c (106 pts). All pts with hormone receptors positive tumor received hormonal treatment according to menopausal status (Pierga et al BCRT 2010). We report here overall survival (OS) and disease free survival (DFS) data at 5 year and associated prognostic factors.

Results: At a median follow up of 49 months, the median DFS was not reached for the whole population and was statistically superior for the HER2 positive cancer patients treated with chemotherapy plus neoadjuvant T compared to the other groups, p = 0.018. The median OS is not reached for the whole population and is statistically higher in HER2 positive tumor group compared to HER2 negative group (p = 0.00077). For 106 HER2 positive breast cancer patients who had received one year of complete trastuzumab treatment, there was no significant difference in OS and DFS between pts who started T in neoadjuvant setting versus in adjuvant setting. DFS and OS were not significantly influenced by pathological Complete Response rate (pCR) (respectively, p = 0.22 and p = 0.56). At multivariate analysis including 6 factors (age, tumor size, clinical lymph node, ER, PgR), factors which influenced OS were tumor size (p = 0.03) and ER expression (p = 0.06) and for DFS, clinical lymph node status (p = 0.049) and PgR expression (p = 0.046).

Conclusion: pCR is not a surrogate of survival in the HER2+subgroup. HER2 positive breast cancer pts receiving trastuzumab have a significant higher OS than those with HER2 negative tumors. OS and DFS do not seem to differ between the neoadjuvant T group and the T adjuvant group.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-14-03.
Abstract P1-14-12: Response to neoadjuvant chemotherapy and prognosis of primary breast cancer according to intrinsic subtype
Authors: Kochi M , Ito M , Ohtani S , Higaki K
Published: August 2014

Background: Response to neoadjuvant chemotherapy of primary breast cancer differs across intrinsic subtypes and is considered as a useful surrogate indicator for prognostic prediction, while some subtype populations seems to take different course.

Methods: We classified 364 primary breast cancer patients, who received neoadjuvant chemotherapy after 2002 at our institute, to 5 intrinsic subtype groups; Luminal A type (LA), Luminal B type (LB), Luminal Her2 type (LH), Her2 type (Her2) and triple negative (TN), according to hormonal sensitivity, Her2 status, and cancer proliferation. We evaluated their pathological response to neoadjuvant chemotherapy by response criteria of Japanese Breast Cancer Society. Pathological complete response (pCR) is defined as Necrosis or disappearance of invasive cancer of breast in this study, approving residual non-invasive component of breast or axillary lymph node metastasis. We also examined disease free survival (DFS) and overall survival (OS) by each subtypes statistically, using Kaplan-Meier method and Log-rank test.

Results: Number of patients in each subtype were as follows; LA: 141 patients, LB: 41 patients, LH: 38 patients, Her2: 54 patients, and TN: 90 patients. Surveillance period is 4–105 months (median: 37 months). Most of cases in all subtypes received both anthracycline and taxane. 27 cases in LH (71.1%) and 48 cases in Her2 (88.9%) were treated with trastzumab. 7 cases in LA (5.0%), 4 cases in LB (9.8%), 9 cases in LH (23.7%), 31 cases in Her2 (57.4%), and 26 cases in TN (28.9%) achieved pCR. pCR rate was significantly high in Her2 and TN (p < 0.05). LA, LB, LH showed no significant differences in DFS and OS between pCR group and non-pCR group (LA; DFS p = 0.929 OS p = 0.663, LB; DFS p = 0.189 OS p = 0.552, LH; DFS p = 0.508 OS p = 0.691). In Her2 group, pCR group tended to show better prognosis, but there were no significant differences (Her2; DFS p = 0.107, OS p = 0.343). In TN group, pCR group showed significantly better prognosis (TN; DFS p = 0.046, OS p = 0.040), which indicates the correlation between response to neoadjuvant chemotherapy and prognosis.

Conclusion: Our results indicate the correlation between response to neoadjuvant chemotherapy and prognosis in TN breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-14-12.
Abstract P1-14-15: Recent experience of neoadjuvant chemotherapy according to breast cancer subtype: experience from a large United Kindgom teaching hospital
Authors: Rattay T , Kaushik M , Ahmed S , Shokuhi S
Published: August 2014

INTRODUCTION: The most recent United Kingdom (NICE, 2009) guidelines recommend neoadjuvant chemotherapy as the most appropriate initial treatment for locally advanced breast cancer followed by mastectomy. Neoadjuvant chemotherapy should also be offered to patients with early breast cancer who are considering breast conserving surgery that is not advisable at presentation. However, the evidence base is still gathering. This study reviews the recent experience of neoadjuvant chemotherapy in a large United Kingdom breast unit according to breast cancer subtype.

METHODS: Retrospective chart review of patients who received neoadjuvant chemotherapy for breast cancer at University Hospitals of Leicester between January 2008 and March 2011. Patients were identified from the unit database. Breast cancers were typed according to immunehistochemical marker profile: luminal A (ER or PR positive, HER2 negative), luminal B (ER or PR positive, HER2 positive), triple negative (ER and PR negative, HER2 negative), and HER2 positive (ER and PR negative, HER2 positive).

RESULTS: Of 2,489 new patients with breast cancer seen during the study period, 153 patients received neoadjuvant chemotherapy. 20 patients had lobular-type cancers, and the remaining 133 patients had ductal- or mixed-type histology. 101 patients had locally advanced disease and 52 patients presented as early breast cancer. 24 of all patients (15 %) achieved complete pathological response. 18 patients (12 %) never proceeded to surgery, eight of whom died during the study period. In total to date, 23 patients (15 %) have died including one death due to complications from chemotherapy. Of 120 patients initially scheduled for mastectomy, the tumor was downsized in 26 patients (22 %) so that they were able to undergo breast conserving surgery (BCS), with margins involved in six patients. The outcome of neoadjuvant chemotherapy according to breast cancer subtype is presented (Tables 1 and 2).

CONCLUSIONS: In our experience, patients receive neoadjuvant chemotherapy for breast cancer for a variety of indications. Compared to our standard population of breast cancer patients, early mortality remains relatively high, particlularly in the hormone receptor negative subtypes. BCS conversion rates were similar across breast cancer subtypes, but BCS was less likely to be successful in the Luminal A group. Luminal A cancers were also significantly less likely to achieve pCR after neoadjuvant chemotherapy. Breast cancer subtype should be taken into account when scheduling patients for neoadjuvant chemotherapy.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-14-15.
Abstract P1-15-02: Febrile neutropenia (FN) risk assessment and granulocyte colony-stimulating factor (G-CSF) guideline adherence in patients with breast cancer – results from a German prospective multicentre observational study (PROTECT)
Authors: Steffens C-C , Eschenburg H , Kurbacher C , Goehler T , Schmidt M , Eustermann H , Schaffrik M , Otremba B
Published: August 2014

Background: FN is a dose-limiting toxicity of chemotherapy which can affect patient (pt) outcomes. EORTC guidelines recommend G-CSF primary prophylaxis when the overall FN risk assessment is ≥20%. We evaluated risk assessment and guideline adherence (prophylaxis in high-risk pts) in clinical practice among breast cancer patients.

Methods: Eligible pts were enrolled consecutively at centres selected based on experience and geographical spread. Key inclusion criteria were diagnosis of solid tumour or lymphoma, physician-assessed overall FN risk ≥10% (chemotherapy risk plus individual risk factors per EORTC guidelines), and planned primary (PPP) or secondary (PSP) prophylaxis with pegfilgrastim. Pegfilgrastim administered >3 days after chemotherapy completion was classified as therapeutic use. The primary objective was to describe the proportion of pts with an investigator-assessed overall FN risk of >20% or 10–20% receiving PPP or PSP. Secondary objectives included evaluating FN incidence and chemotherapy dose reductions/delays. Data from breast cancer patients only are reported, and prophylaxis in high-risk patients was determined to evaluate guideline adherence.

Results: 1003 pts were enrolled from Nov 2007 to Sept 2010. Mean (±SD) age was 54.4 (±11.2) years, almost all pts (99%) were female. Treatment intent was deemed curative by the investigator in 91% of pts. The investigator-assessed FN risk was >20% in 505 (50%) pts and 10–20% in 414 (41%) pts. Despite eligibility criteria, 84 (8%) pts were assessed by investigators as <10% risk; reasons for inclusion of low-risk pts were not documented. Measured baseline patient risk factors were similar between PPP and PSP pts, and PPP and PSP pts were equally likely to receive chemotherapy with curative intent (92% vs 89%) and less likely to have received prior chemotherapy (11% vs 20%). 100 pts (10%) experienced FN, 43/680 (6%) in the PPP and 53/254 (21%) in the PSP group. 45 (4%) pts experienced FN-related dose reductions or dose delays, with 22/680 (3%) in the PPP group and 22/254 (9%) in the PSP group. In the subgroup of pts assessed as high FN risk, PPP was administered to fewer pts than planned and PSP to more pts than planned (Table).

Conclusion: Adherence to G-CSF guidelines, and reasons for differences between planned and administered prophylaxis warrants further investigation. Comparison of outcomes between prophylaxis groups should be interpreted with caution; however, FN incidence remained low with pegfilgrastim PP among breast cancer pts in German clinical practice.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-15-02.
Abstract P2-01-04: Long term independent prognostic impact of circulating tumor cells detected before neoadjuvant chemotherapy in non-metastatic breast cancer: 70 months analysis of the REMAGUS02 study
Authors: Bidard F-C , Delaloge S , Giacchetti S , Brain E , de Cremoux P , Vincent-Salomon A , Marty M , Pierga J-Y
Published: August 2014

Background: Circulating tumor cells (CTCs) isolated by CellSearch® from peripheral blood of metastatic breast patients have been shown as strong independent prognostic factor for progression-free and overall survival. With this technique, the REMAGUS02 prospective multicentric study was the first to report that CTC detection (≥1CTC/7.5ml) before and/or after neoadjuvant chemotherapy was independently associated with distant metastasis-free survival (DMFS, 18 months follow-up, Pierga CCR 2008) and with overall survival (OS, 36 months follow-up, Bidard Ann Oncol 2010).

Patients and Methods: In 115 non-metastatic patients diagnosed with large operable or locally advanced breast cancer, we prospectively detected CTC using the CellSearch system before and after neoadjuvant chemotherapy in the REMAGUS02 trial (Pierga, BCRT 2010). For this report, survival analyses were performed at a median follow-up of 70 months.

Results: After a median follow-up of 70 months, 20 distant metastatic relapses and 14 deaths have been observed among the 115 patients included. CTC detection before chemotherapy (in 23% of patients) is an independent prognostic factor for both DMFS [p = 0.03, relative risk (RR)=3.2] and OS [p = 0.05, RR = 3.7] in multivariate analyses, together with triple negative tumor status. At long-term, CTC detection after chemotherapy (17%) had no clear prognostic significance (p = 0.15 and 0.22, respectively). Complete pathological response as well as tumour size and all other variables tested did not appear as predictive in this model.

Conclusions: Baseline CTC detection is a new independent prognostic factor in the neoadjuvant setting, and is, in this trial, superior to pathological tumor response to predict the survival of non-metastatic breast cancer patients. We confirm therefore our previous results reported with shorter follow-up.

Supported by PHRC AOM/2OO2/02117, Pfizer inc., Roche, sanofi-aventis. ISRCTN10059974

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-04.
Abstract P2-01-08: Different numbers and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes.
Authors: Peeters DJ , van Dam P-J , Wuyts H , Van den Eynden GG , Jeuris K , Prové A , Rutten A , Peeters M , Pauwels P , Van Laere SJ , Hauspy J , van Dam PA , Vermeulen PB , Dirix LY
Published: August 2014

Introduction: The enumeration of circulating tumour cells (CTCs) with the EPCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). However, breast cancer has been shown to be a molecularly heterogeneous disease. The aim of this study was to assess potential differences in the detection and prognostic significance of CTCs according to the immunohistochemically defined molecular subtypes of breast cancer.

Methods: CellSearch CTC counts were obtained from 110 patients with MBC prior to first line systemic treatment, treated at GZA Hospitals Sint-Augustinus between november 2007 and december 2011. Clinicopathological variables were prospectively entered in a database. Based on the St-Gallen surrogate definitions of intrinsic breast cancer subtypes (Goldhirsch et al. Ann Oncol 2011), patients were divided in 5 groups: luminal A (ER/PR+, HER2−, Bloom-Richardson histological grade I-II), luminal B – HER2 negative (ER/PR+, Her2−, grade III), luminal B – HER2 positive (ER/PR+, HER2+, any grade), HER2 positive – non luminal (ER/PR−, HER2+), and triple negative (TN) (ER/PR−, HER2−). Differences in progression free survival (PFS) and overall survival (OS) according to the FDA approved prognostic cut-off of ≥5 CTC/7.5 ml blood were estimated using Kaplan Meier and Cox proportional hazard statistics.

Results: CTC were detected in 78 of 110 (71%) patients. Higher detection rates and numbers of CTC were observed in patients with luminal A and TN breast cancer as compared to patients with luminal B and HER2 positive disease. However, no differences in positivity rates were observed between molecular subtypes according to the 5 CTC prognostic cut-off point (table 1). After a median FU time of 3.1 years, 39 patients had died. In the total study population, the presence of ≥5 CTC was an independent predictor of PFS and OS in multivariate analysis (PFS: HRCTC≥5=2.236 (1.366–3.658), p = 0.001; OS: HRCTC≥5=3.180 (1.553–6.509), p = 0.002). When analyzing subgroups separately, a lower prognostic power was observed in the HER2 positive and luminal B subgroups.

Conclusion: Significant differences were observed in the detection and prognostic significance of EPCAM positive CTC according to the immunohistochemically defined breast cancer subtypes. Interestingly, CTC were detected more frequently in patients with luminal A and TN tumors. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2 positive patients with MBC. Our data independently confirm those reported by Giordano et al. (Ann Oncol 2010) in a large clinically uniform population of patients with MBC before the start of first-line treatment.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-08.
Abstract P2-04-03: A robust transgenic mouse model to study male breast cancer.
Authors: Krause S , Lurvey HL , Tobin H , Ingber DE
Published: August 2014

Male breast cancer accounts for about 1% of all breast cancers and even though it is a rare disease, the incidence rate has increased steadily during the past 20 years. Study of the male form of the disease has been hampered by a lack of relevant animal models as it is commonly believed that male mammary glands do not contain ducts. Female FVB C3(1)-SV40Tag transgenic mice spontaneously develop hyperplastic mammary lesions that progress to invasive mammary tumors, whereas most of the transgenic males develop prostatic hyperplasia that progresses to adenocarcinoma. However, upon closer examination, we discovered that approximately 45% of transgenic males develop mammary tumors by 40 weeks of age, often prior to developing prostate tumors. The course of male mammary development is similar to that observed in the females but with a later onset. Hyperplastic ducts form as early as 15 weeks of age and progress into DCIS-resembling tumors before developing into invasive tumors that invade locally into adipose tissue and muscle and occasionally form metastases to lung and other organs. We then examined male mammary development in the FVB background strain (wild type) and showed that all males develop mammary ducts that continue to grow and branch throughout adulthood. The morphology of these ducts was similar to that of females with a central lumen surrounded by concentric layers of luminal epithelium, myoepithelium, a basement membrane and surrounding stroma. A study of CD1 male mice confirmed that male mammary development was not specific to the FVB strain but rather, CD1 males showed similar mammary ductal development. Male mammary development in FVB wild type animals and tumor progression in the transgenic males was characterized in terms of cell proliferation, apoptosis and expression of various markers of differentiation including hormone receptor status. In summary, our results demonstrate that FVB C3(1)-SV40Tag transgenic males represent a highly robust and reproducible model for the study male breast cancer. Our long-term goal is to leverage the information we uncovered through this effort to design novel therapies for breast cancer treatment in men.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-03.
Abstract P2-04-06: Transgenic expression of a breast-cancer specific GATA3 mutant leads to mammary hyperplasia
Authors: Kenny PA , Chandiramani N
Published: August 2014

GATA3 is expressed in normal luminal breast epithelial cells and is a key transcription factor in mammary gland development. In human breast tumors, high GATA3 protein levels are associated with estrogen receptor (ER) positivity and low grade. Recent published reports indicate that somatic mutations of GATA3 occur in up to 21% of ER+ tumors. These authors suggest, but do not demonstrate, that these GATA3 mutations are loss-of-function null alleles and that GATA3 is a haploinsufficient tumor suppressor gene.

The published mutational data indicate a striking enrichment of GATA3 mutations in exons 5 and 6 (amino acids 309–443) which encode the second of two DNA binding zinc fingers. This suggests that mutant proteins lacking this zinc finger may confer a survival advantage on breast cancer cells. Given this apparently strong selective pressure for mutations in this hotspot, it seems unlikely that these mutations are loss-of-function alleles.

We explored this directly by generating a MMTV-GATA3mutant mouse which specifically expresses a mutant GATA3 protein in the mammary gland. Non-parous pubertal transgenic mice exhibit a pronounced mammary hyperplasia and have increased ductal branching compared to wildtype littermates. These data confirm that the mutant GATA3 mutations commonly found in breast cancer are not null alleles, but that they instead have a gain-of-function phenotype. We are currently evaluating the effect of mutant GATA3 on the kinetics of mammary tumorigenesis by crossing these mice to the MMTV-PyMT model.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-06.
Abstract P2-05-07: Comparison of hormone receptor and HER-2 expression in primary breast cancers and sentinel lymph node metastases
Authors: Rokutanda N , Horiguchi J , Takata D , Nagaoka R , Sato A , Tokiniwa H , Tozuka K , Uchida S , Takeyoshi I
Published: August 2014

Background: Recently, it is a hot topic that the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor type 2 (HER2) may differ between the primary tumors and the paired recurrent metastatic tumors. On the other hand, the concordance of the ER, PR, and HER2 status between the primary tumor and ipsilateral axillary lymph node metastases is reported to be high in previous study. However, in these reports, the majority of lymph nodes metastases examined are macrometastases obtained from axillary lymph node dissection. The aim of the present study is to compare the ER, PR, and HER2 status of the primary breast tumors with that of sentinel lymph node micrometastases and macrometastases.

Methods: A total of 770 breast cancer patients underwent sentinel lymph node biopsy between 2006 and 2010 at our hospital. Among them, 87 patients who had only one node positive were eligible for our study. ER, PR, and HER2 status were determined by immunohistochemistry (IHC) and/or FISH.

Result: We analyzed 76 patients with 50(66%) patients with macrometastasesn and 26 (34%) micrometastases, except 11 cases those were indeterminate for IHC. ER status of primary tumor and the paired metastatic lymph node were almost full concordance except one case, in which ER was negative in primary tumor but ER was positive in lymph node. Discordance for PR was 15.8% (n = 12). Among these, six patients had PR-positive on primary and PR-negative in lymph node while six patients had PR-negative in primary and PR-positive in lymph node. Discordance for HER2 between primary tumor and metastatic lymph node was 5.3% (n = 4). Among these, three had negative in primaries and positive in lymph nodes while one had positive in primary and negative in lymph node. A discordance between primary and lymph node in HER2, ER, or PR status was observed in 14 of 67 (18.4%) cases.

Conclusions: The concordance in HER2, ER, and PR was high between the primary tumor and sentinel lymph nodes. However some cases had discordance of receptor status and these result may cause of discordance for distant metastases or poor prognosis.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-07.
Abstract P2-05-11: Proteomic identification and in-silico verification of subtype-specific signatures in breast cancer.
Authors: Pavlou MP , Dimitromanolakis A , Diamandis EP
Published: August 2014

Breast cancer is a highly heterogeneous disease with different subtypes presenting distinct clinical characteristics. During the last decade has been shown that molecular classification of breast cancer holds the promise for the development of novel prognostic tools and the identification of novel treatment targets. However, proteins are the main mediators of biological processes and the molecular targets of the majority of drugs. Moreover, the proteome integrates the cellular genetic information and environmental influences. Hence coupling proteomic and transcriptomic studies may augment the development of targeted therapies and the identification of disease-relevant proteins networks.

Here, we report an extensive mass spectrometry-based (MS) secretome analysis of eight breast cancer cell lines representing, the three main breast cancer subtypes (luminal, basal and HER2-neu amplified) in search of subtype-specific proteomic signatures. Following a bottom-up proteomic approach coupled to tandem mass spectrometry the conditioned media of 8 breast cancer cell lines were analyzed. More than 5,200 non-redundant proteins were identified in the conditioned media of the 8 cell lines with a false positive rate less than 1%. The mass spectrometry-based identification of 5 proteins was successfully verified by ELISA.

For the generation of subtype-specific proteomic signatures, proteins common to all cell lines of the same subtype but not present in the other two subtypes were identified. Twenty-three, four and four proteins were found uniquely in basal, HER2-neu amplified and luminal breast cancer cells. Notably, ERBB2 was one of the proteins uniquely identified in the HER2-neu amplified subtype verifying the validity of our approach.

Given that most of these proteins have no quantitative methods available at present, we opted to preliminarily examine the relationship of these candidates with breast cancer subtypes by using an in silico approach, based on transcriptomic data. We performed an in silico mRNA expression analysis using publicly available data from four independent experiments containing a total of 1039 patients with primary breast cancer. The expression profiles of 15 of 31 proteins investigated showed significant correlation with estrogen receptor expression and power to distinguish subtypes. The development of multiplex quantitative mass spectrometry-based assays for the quantification of the proteomic signatures in relevant biological specimens along with immunohistochemical studies for two of the 31 candidates are currently ongoing.

The significance of these subtype-specific proteins in breast cancer prognosis and disease progression warrants further investigation.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-11.
Abstract P2-05-14: Clinical significance of Lysil oxidase-like 2 (LOXL 2) in breast cancer
Authors: Ahn SG , Lee HM , Hwang SH , Lee SA , Jeong J , Lee H-E
Published: August 2014

Introduction: Several preclinical studies provided relevant evidences that lysine oxidase-like 2 (LOXL2) is associated with invasiveness and metastasis in breast cancer. To investigate the clinical significance of LOXL2, we performed survival analysis with LOXL-2 in breast cancer patients.

Method: A cohort treated at Gangnam Severance hospital, Seoul, Korea between January 1996 and December 2004 was studied. The immunohistochemical analyses with LOXL-2 antibody were performed in the 309 patients. The patients was stratified into two groups based on the expression level of LOXL-2. Kaplan-Meier and Cox's methods were used to define prognostic factors associated with breast cancer survival.

Results: LOXL-2 positive group was 50 patients (16.2%) and 259 patients (83.8%) are LOXL-2 negative group. The proportion of estrogen receptor-negative patients was significantly higher in LOXL-2 positive group (54.0 vs. 37.0 %, P = 0.040). The Kaplan-Meier overall survival estimate at 5 year for the breast cancer patients with negative LOXL-2 was higher than that for the patients with positive LOXL-2 (88.0 vs. 71.8 %, P = 0.008). In the multivariate analysis for overall survival, lymph node metastasis (Hazard Ratio (HR) 0.32, 95% confidence interval (CI) 0.17–0.61) and positive LOXL-2 (HR 0.53, 95% CI 0.29–0.97) were significantly associated. In the multivariate analysis for distant metastasis-free survival, age younger than 35 years (HR 0.52, 95% CI 0.30–0.91), lymph node metastasis (HR 0.29, 95% CI 0.16–0.54), high histologic grade (HR 0.54, 95% CI 0.32–0.89) and positive LOXL-2 (HR 0.55, 95% CI 0.31–0.97) were demonstrated as significant prognostic factors.

Conclusion: In breast cancer patients, overexpression of LOXL-2 is a poor prognostic factor for overall survival and distant metastasis-free survival.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-14.
Abstract P2-05-19: Pathway-based analysis of breast cancer
Authors: Song D , Cui M , Fan Z , Yang Y , Xue L , Zhang DY , Ye F
Published: August 2014

Objective: Although HER2 and ER pathways are predominant pathways altered in breast cancer, it is now well accepted that many other signaling pathways are also involved in the pathogenesis of breast cancer, especially in triple negative breast cancer. The understanding of these additional pathways may assist in identifying new therapeutic approaches for breast cancer.

Methods: 13 invasive ductal carcinoma tissues and 5 benign breast tissues were analyzed for the mRNA expression level of 1243 cancer pathway-related genes using SmartChip (WaferGen, CA), a real-time PCR-base method. In addition, the levels of 154 cancer pathway-related proteins and phosphoproteins were measured using our innovative Protein Pathway Array.

Results: Out of 1,243 mRNAs, 68.7% were detected in breast cancer and 73 mRNAs were statistically significant between benign and cancer tissues. Of these mRNAs, 105 only expressed in breast cancer tissues and 33 mRNAs only expressed in normal breast tissues. Out of 154 proteins and phosphoproteins, 39% were detected in cancer tissues and 50 proteins were significantly differentiated between tumor and normal tissues. Interestingly, only 3 genes (CDK6, Vimentin and SLUG) showed decrease of both protein and mRNA. Six proteins (BCL6, CCNE1, PCNA, PDK1, SRC and XIAP) showed differentially expressed between tumor and normal tissues but no differences were observed at mRNA levels. Analyses of mRNA and protein data using Ingenuity Pathway Analysis showed more 15 pathways were altered in breast cancers and 6 of them shared between mRNAs and proteins, including p53, IL17, HGF, NGF, PTEN and PI3K/AKT.

Conclusion: There is a broad dysregulation of various pathways in breast cancer both at protein levels and mRNA levels. It is important to note that mRNA expression does not correlate with protein level, suggesting that different regulation mechanisms between proteins and mRNAs.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-19.
Abstract P2-10-20: A tumor DNA complexity index is an independent predictor of survival in a dataset of 1950 breast cancers; a METABRIC group study.
Authors: Vollan HKM , Rueda OM , Børresen-Dale A-L , Aparicio S , Caldas C
Published: August 2014

Background: Cancer genomes undergo substantial alterations during carcinogenesis. Patterns of such rearrangements differ with respect to cancer subtypes and may affect crucial parts of the genome. DNA based markers would make clinical implementation more feasible as markers based on RNA expression are challenging due to the intrinsic problem of RNA stability and volatility in expression patterns. Complex genomic rearrangements in breast cancer genomes, quantified by the complex arm-wise aberration index (CAAI), have previously been shown to be of prognostic value. However this finding has not been validated in independent large-scale breast cancer cohorts or compared with other molecular predictors.

Methods: A total of 1950 breast carcinomas from the METABRIC cohort with log term follow up were included in this study. Microarray based copy number and expression data from fresh frozen primary tumors were available. Cases were classified as CAAI positive if at least one complex event was present in the tumor genome. The clinical end points were breast cancer specific survival (BCSS) and overall survival (OS).

Results: A total of 33 % of breast cancer cases were CAAI positive with a Cox proportional hazard ratio (HR) of 1.78 (95% CI, 1.49–2.12) for BCSS. Using multivariate Cox regression the adjusted HR for BCSS of 1.42 (95% CI, 1.12–1.80) and 1.44 (95% CI, 1.02–2.03) in estrogen receptor positive (ER+) and estrogen receptor negative breast cancers, respectively. Gene expression classifiers based on OncotypeDX and MammaPrint were not independently prognostic in the ER− subset. Significant difference in survival was both seen in patients that received systemic treatment, and in patients that did not.

Conclusions: DNA based prognostic profiling is a robust method for both fresh frozen and FFPE tumor material. This study shows that complex genomic alterations of tumor DNA, measured as CAAI, is an independent predictor of survival in breast cancer, in both ER+ and ER− disease in a large scale breast cancer cohort with long term follow up. The effect seems unrelated to systemic endocrine treatment or chemotherapy.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-20.