bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P4-11-03: Effect of tamoxifen and raloxifene on proliferative activity of breast epithelium in premenopausal women
Authors: Freitas-Junior R , Lucato MT , Moreira MA , Bernardes-Junior JM , Pinto SA , Paulinelli RR , Soares LR
Published: August 2014

Background: Both Tamoxifen and raloxifene have been demonstrated to be effective to reduce the incidence of breast cancer among women. Objective: To compare the effects of raloxifene (60 mg/day) and tamoxifen (20 mg/day) on the proliferative activity of normal human breast epithelium, during the luteal phase of the menstrual cycle, by means of the immunoexpression of Ki-67/MIB-1. Patients and Methods: Forty-eight women presenting benign breast nodules with an indication for lesion excision took part in this study. These women were randomized to use tamoxifen or raloxifene for 22 days. They underwent surgery after using the medication, and a one-centimeter fragment of normal breast tissue was removed to study the immunoexpression of Ki-67. For this, the frequency of cells immunolabeled for Ki-67/MIB-1 per thousand epithelial cells was quantified. Results: The mean percentage ratios between immunolabeled and non-labeled cells were 2.02 (±1.09) and 3.13 (±3.23) for the tamoxifen and raloxifene groups, respectively. There was no statistically significant difference between the two groups, tamoxifen (n = 16) and raloxifene (n = 14), in relation to the immunoexpression of Ki-67 (p = 0.205). The mean of progesterone level was 19.62 (±17.67) for the tamoxifen group and 14.59 (±8.98) for the raloxefene group (p = 0.32). The plasma levels of estradiol, the values found were: 355.72 (± 337.98) and 171.02 (± 119.83) for groups of tamoxifen e raloxifene groups, respectively (p = 0.055). Conclusion: In this study, we observed that there was no difference in the potential for reducing the proliferative activity of normal breast epithelium between tamoxifen and raloxifene in premenopausal women.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-11-03.
Abstract P4-12-12: Phase 2, multicenter, single-arm study of eribulin mesylate + trastuzumab as first-line therapy for locally recurrent or metastatic HER2-positive breast cancer
Authors: Wilks S , Puhalla S , O'Shaughnessy J , Schwartzberg L , Berrak E , Song J , Rege J , Cox D , Vahdat L
Published: August 2014

Background: Eribulin mesylate, a non-taxane microtubule dynamics inhibitor, has been approved for patients with metastatic breast cancer (MBC) who have previously received ≥2 chemotherapeutic regimens for MBC. We present final data from a phase 2 study that evaluated efficacy and safety of eribulin + trastuzumab as first-line therapy for locally recurrent or metastatic human epidermal growth factor receptor 2 positive (HER2+) BC.

Methods: Patients received eribulin mesylate at 1.4 mg/m2 IV on days 1 and 8 of each 21-day cycle and an initial trastuzumab dose of 8 mg/kg IV on day 1, followed by 6 mg/kg on day 1 of each subsequent cycle. Endpoints include objective response rate (ORR), safety, progression-free survival (PFS), time to response (TTR), and duration of response (DOR). Tumor assessments were conducted every 6 weeks for the first 6 cycles and every 6-12 weeks thereafter per RECIST 1.1.

Results: Fifty-two patients with a median age of 60 years (range 31- 81) were treated; 96% had stage IV disease, 73% had visceral disease, and 48% had liver metastases. Thirty one patients had prior neo/adjuvant chemotherapy (11 had prior anthracycline, and 22 had prior taxane). Patients received a median of 10(0, 33) cycles of eribulin and 11(1, 31) cycles of trastuzumab. ORR was 67% with median TTR of 1.3 months and PFS of 11.5 months (Table). The most common (>5%) Grade 3/4 treatment related treatment emergent (TRTE) AEs were neutropenia (n = 20; 38.5%), peripheral neuropathy (n = 14; 26.9%) and febrile neutropenia (n = 4; 7.7%). Serious TRTE AEs occurred in 14 patients and included neutropenia (n = 9; 17.3%), febrile neutropenia (n = 4; 7.7%), and peripheral neuropathy (n = 3; 5.8%). Reasons for discontinuation were AEs (n = 7) and PD (n = 22).

Table. Summary of Efficacy EndpointsEfficacy EndpointsEribulin/Trastuzumab N = 52Objective Response Rate, n (%)35 (67)- Complete Response (CR)2 (4)- Partial Response (PR)33 (64)Stable Disease (SD)15 (29)Progressive Disease (PD)1 (2)Not Evaluable1 (2)Overall Clinical Benefit Rate, n (%)42 (81)Time to First Objective Response, median months (95% CI)1.3 (1.2, 1.4)Duration of Objective Response, median months (95% CI)a11.1 (6.5, 17.8)Progression-Free Survival, median months (95% CI)11.5 (7.3, 13.5)Duration of Stable Disease, median months (95% CI)7.1 (5.2, 13.5)Clinical Benefit Rate = ORR+ ≥6 mo SD; an = 35

Conclusions: This study suggests that the combination of eribulin + trastuzumab first-line therapy for locally recurrent or metastatic HER2+ BC has an acceptable safety profile and results in considerable tumor response with a long DOR. Additional larger studies with this combination are warranted.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-12.
Abstract P4-12-23: Subcutaneous trastuzumab (Herceptin™) in patients with HER-2 positive early breast cancer: A UK time and motion study in comparison with intravenous formulation
Authors: Burcombe R , Chan S , Simcock R , Samanta K , Percival F , Barrett-Lee P
Published: August 2014

Aim:

To quantify active healthcare professional (HCP) time and costs associated with subcutaneous (SC) and intravenous (IV) infusion administration of trastuzumab (Herceptin) in the treatment of patients with HER2-positive early breast cancer within the adjuvant PrefHer trial; secondly, to measure patient time in the care unit and patient chair time for both routes of administration.

Methods:

A UK multi-centre prospective, observational time and motion study was conducted alongside the PrefHer trial (ClinicalTrials.gov id: NCT01401166). Trained observers measured the duration of each SC and IV related task HCPs undertook and recorded patient time in the chemotherapy unit and treatment chair. The type and quantity of medical consumables used with each route of administration were also collected. 24 patient episodes were recorded (12 SC, 12 IV). Mean total administration time was calculated as the mean sum of task times, for both IV and SC formulations. The mean cost of each route of administration was calculated as the mean cost of HCP time plus the mean cost of consumables used. HCP time was costed using data from the Personal Social Services Research Unit. Consumables were costed using hospital pharmacy data and online sources.

Results:

Mean active HCP time for IV preparation and administration was 92.6 minutes compared with 24.6 minutes for SC administration. The mean cost for IV preparation and administration was £144.96 (£132.05 of HCP time and £12.92 of consumables) versus £33.15 (£31.99 of HCP time and £1.17 of consumables) for SC administration. Mean time spent by patients in the care unit and treatment chair was 94.5 minutes and 75 minutes respectively for IV, and 30.3 minutes and 19.8 minutes for SC.

SC administration of trastuzumab could translate to a HCP time saving of 68 minutes (34.5 minutes of preparation time and 33.5 minutes of administration time) (versus IV) with a total cost saving of £111.81 per patient episode. This equates to a potential saving of £2012.58 over a full course of adjuvant trastuzumab treatment (18 cycles).

Conclusion:

Substituting IV infusion with SC administration of trastuzumab may lead to a substantial reduction in active HCP time, consumable use and overall cost. The reduced patient chair and unit time could provide increased capacity within existing resources.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-23.
Abstract P4-16-07: Mesenchymal-epithelial transition (MET) and epithelial-mesenchymal transition (EMT) are closely related to chemotherapy resistance of triple negative breast cancer
Authors: Deng X , He J , Chang HR
Published: August 2014

Triple negative breast cancer (TNBC) is among the most aggressive clinical phenotypes and the presence of residual resistant cancer cells of TNBC after chemotherapy is associated with tumor recurrence and metastasis. In recent years more and more evidence highlight a crucial role of CD44 (+)/CD24 (-/low) (stem/progenitor cell-phenotype) population in cancer drug resistance but the mechanism remains elusive. In the current study, we screened for CD44 and CD24 using Fluorescence-activated cell sorting (FACS) in 8 TNBC cell lines before and after Docetaxel and Doxorubicin treatment. Our data showed that after Docetaxel treatment, several CD44 (+)/CD24 (-/low) cell lines changed their phenotypes to CD44 (+)/CD24 (+) and most of the CD44 (+)/CD24 (+) cell lines kept their double positive phenotypes. In contrast, after Doxorubicin treatment, all the CD44 (+)/CD24 (-/low) cell lines kept their phenotypes and all the CD44 (+)/CD24 (+) cell lines changed their phenotypes to CD44 (+)/CD24 (-/low). Furthermore, among the studied 8 cell lines, CD44 (+)/CD24 (+) cell population is always more resistant to Docetaxel and CD44 (+)/CD24 (-/low) population is always more resistant to Doxorubicin. Our finding of EMT transition from CD44 (+)/CD24 (+) to CD44 (+)/CD24 (-/low) in breast cancer cell lines and its inducible by Doxorubicin is consistent with the literature. However, our results showed for the first time that both EMT and the inverse MET in TNBC could be induced by specific chemotherapy drugs based on the phenotypes of CD44 and CD24. Besides, our gene microarray data also showed that TGFb2, BCL2, KLF6 and TAF4 pathways were dramatically changed in both CD44 (+)/CD24 (+) to CD44 (+)/CD24 (-/low) transition and CD44 (+)/CD24 (-/low) to CD44 (+)/CD24 (+) transition. Further study of these pathways in the transitions may give us new clues to overcome the chemotherapy resistance and lead us to develop successful therapeutic strategies of different types of TNBC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-07.
Abstract P4-17-02: Guidelines for the definitions of time-to-event endpoints in randomized clinical trials: Results of the datecan project for breast group
Authors: Gourgou-Bourgade S , Dabakuyo-Yonli S , Cameron D , Cardoso F , Poortmans P , Asselain B , Azria D ,   For DATECAN Group
Published: August 2014

Background: Surrogate endpoints of overall survival (OS) are increasingly being used in cancer RCTs with the necessity of reducing randomized clinical trial (RCT) duration, cost and number of patients. However, most of these endpoints currently lack of standardized definition enabling a comparison of RCT results. The objective of the Definition for the Assessment of Time-to-event Endpoints in CANcer trials (DATECAN) initiative is to provide guidelines to standardize definitions of time-to-event endpoints in RCTs for different cancer sites. Some recommendations have been proposed for specific cancer sites but they do not rely on a formal consensus methodology. Here, we presented results for BREAST cancer.

Methods: We relied on a validated formalized consensus process for the development of practice guidelines using a modified Delphi consensus method. International experts with various backgrounds and expertises were involved. First, the coordinating committee, a group of statisticians and epidemiologists involved in the design and conduct of RCTs, led a comprehensive literature review to identify time-to-event endpoints, events of interest and the existence of guidelines in metastatic and non metastatic settings. The steering committee, which included additional medical experts, validated the list and prepared the questionnaire sent for rating to an independent expert committee.

Results: The consensus process involved 2 rounds of scoring (31 experts) and finally one in-person meeting. Each expert had to rate on a 1-9 scale if s/he agreed or not for including events (e.g. death from breast cancer) in the definition of time-to-event endpoints (e.g. progression-free survival). 150 events had to be scored for the 11 selected endpoints. Consensus was reached for 57% of the events after the 2 rounds of scoring. After the in-person meeting, consensus was reached for all the remaining events but one.

Conclusions: The DATECAN guidelines should help standardizing definitions of commonly used endpoints. This process should (i) facilitate the comparison of RCTs and (ii) improve the quality of future RCTs by providing better estimation of sample size and treatment effect by disseminating the recommendations in major congress. (previously presented to ASCO’13)

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-17-02.
Abstract P5-04-06: Loss of the E3 ubiquitin ligase HACE1 plays a critical role in transformation of mammary cells and clinical progression of human breast cancer via accumulation of active Rac1
Authors: Goka ET , Lippman ME
Published: August 2014

Invasive ductal breast cancer arises when the immediate precursor, ductal carcinoma in situ (DCIS), breaches the basement membrane of the ductal lumen and invades into the surrounding tissue. Although this transition has been implicated as a crucial step in the progression of breast cancer, the specific alterations responsible for this transition have yet to be identified.

HER2/neu, a member of the epidermal growth family receptor (EGFR) family, is frequently overexpressed in non-invasive lesions (50-60%) but is significantly less common in invasive breast cancer (20-30%). This suggests that while HER2/neu may be partially transformative, additional alterations are required for mammary epithelial cells to achieve full malignant transformation.

To identify novel genes capable of cooperating with HER2, we performed an insertional mutagenesis screen for genes whose alteration induces anchorage-independent growth on primary mouse mammary epithelial cells isolated from MMTV-neu transgenic mice. We identified HECT domain and ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) as a putative breast tumor suppressor gene whose loss contributes to the transformative process.

Loss of HACE1 expression is commonly seen in publicly available breast cancer patient data sets as well as in established breast cancer cell lines supporting the role of HACE1 as a breast cancer tumor suppressor gene. Knockdown of HACE1 in HER2 overexpressing human mammary epithelial cells (HMECs) enhanced colony formation over HER2 overexpressing HMEC controls cells. Moreover, knockdown of HACE1 alone was sufficient to allow anchorage-independent growth in soft agar in the HMECs while the overexpression of HACE1 in breast cancer cell lines diminishes clonogenic capacity in soft agar.

We confirmed recent studies that have shown that HACE1 is capable of tagging the Rho GTPase Rac1 for ubiquitin-mediated proteasomal degradation. In mammary epithelial cells, the loss of HACE1 leads to enhanced levels of active Rac1 resulting in increased clonogenicity, migration and invasion. Importantly, we show that targeting Rac1 can rescue the effects of HACE1 loss in mammary epithelial cells. Our results establish HACE1 as a breast cancer tumor suppressor gene by attenuating active Rac1 signaling. Our work supports the role of Rac1 as a critical signaling node in breast cancer and that loss of HACE1 leads to enhanced Rac1 signaling resulting in driving cancer progression. Furthermore, our results suggest the role of HACE1 loss as a biomarker for tumor progression and may identify patients vulnerable to Rac1 or Rac effector targeted therapies.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-06.
Abstract P5-04-18: Identification of a novel combination therapy that prevents the metastatic outgrowth and reduces the viability of dormant breast cancer cells: implications for clinical translation
Authors: El Touny LH , Vieira A , Mendoza A , Khanna C , Hoenerhoff MJ , Green JE
Published: August 2014

Recurrent metastatic disease many years after initial therapy is a major cause of breast cancer (BC) mortality and strongly suggests that disseminated cells survive long periods in a growth-arrested state that is refractory to chemotherapy, known as “cellular dormancy.” Alterations in the components of the extracellular matrix (ECM), such as fibrotic foci and collagen-1 (Col-1) deposition, are associated with more aggressive breast cancer, increased metastasis and poor prognosis for BC patients. This suggests that ECM alterations may have a role in influencing the behavior of dormant BC cells. In fact, we have previously shown both in vitro (using 3-dimensional culture) and in vivo (preclinical model of fibrosis at the lung metastatic site) that alterations in the ECM surrounding disseminated, dormant BC cells can induce signaling pathways requiring Src and ERK1/2 activation that switch dormant cells into a proliferative state. We now demonstrate using the Src inhibitor AD0530 or Src shRNA, that the proliferative outbreak of dormant BC cells in Col-1-enriched microenvironments is prevented through the induction of a reversible G1-phase arrest and the upregulation of nuclear cyclin dependent kinase inhibitor p27Kip1, without affecting the viability of the dormant cells. Src inhibition, however, is not efficacious in regressing already established metastatic lesions, suggesting that Src signaling is crucial to engage the dormant-to-proliferative switch but is not necessary for survival once the cells have begun to proliferate. Since ERK1/2 activation is also required for the dormant-to-proliferative switch, we determined the effect of the MEK1/2 inhibitor AZD6244 that prevents ERK1/2 activation on the proliferative switch alone and in combination with the Src inhibitor. Importantly, we show that combination treatment with AZD0530 and the MEK1/2 inhibitor AZD6244 of dormant cells undergoing the dormant-to-proliferative switch induces apoptotic cell death in a significant fraction of the dormant cells, which is not seen with either inhibitor alone, and significantly delays metastatic outgrowth in the lung metastatic site. In conclusion, inhibition of Src alone prevents the proliferative response of dormant cells to external stimuli without affecting survival, but the addition of a MEK1/2 inhibitor suppresses survival of dormant cells, indicating that this combination may have clinical value in preventing breast cancer recurrence.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-18.
Abstract P5-05-07: Promestriene effects on estrogen-sensitive breast cancer cell proliferation in vitro
Authors: Almodovar AJO , Litherland SA , Courtneidge S , Decker DA
Published: August 2014

Introduction: Promestriene (3-propyl ethyl, 17B-methyl estradiol) is a synthetic estrogen analogue with potential as a topical treatment for vaginal atrophy caused by estrogen deprivation. Topical Promestriene is minimally absorbed with chronic use and has been reported to significantly improve the symptoms of vaginal atrophy. However, to be considered for use for these symptoms in estrogen responsive post-menopausal breast cancer survivors on an aromatase inhibitor, Promestriene would need to show none of the cyto-proliferative properties of estrogen.

Methods: We conducted in vitro testing of estrogen responsive breast cancer cell line MCF-7, T-47D, and BT-474 for dose-dependent, estrogen-like functional responses to Promestriene in both estrogen-rich and estrogen-deprived conditions. As assay controls, cultures treated with Fulvestrant, Testosterone, Progesterone and Estradiol were analyzed in parallel.

Analysis: Dose dependent Estrogen-like responses in the cell lines were measured as 1) proliferation using flow cytometric analysis of CSFE fluorescence, 2) estrogen receptor expression by flow cytometry, and 3) GREB 1 RNA expression using TaqMan real time PCR quantitative analysis. All cultures were grown to quiescence in RPMI medium supplemented with 10% fetal calf serum. To induce estrogen deprivation, half of the cultures were then held 3 days in media pre-treated and containing anti-estrogen specific antibodies to sequester any residual estrogen from the culture. Data for each culture treatment were analyzed using linear regression analysis and compared using multifactorial or pairwise analyses to determine the significance of estrogen-like responses relative to the estradiol and untreated controls.

Results: In estrogen sufficient cultures, Promestriene exhibited no cell proliferative properties on MCF-7, BT-474, or T47-D tumor cells, even at very high concentrations. However, when estrogen in the media was sequestered and culture allowed to quiesce without estrogen receptor stimulation, low dose Promestriene effects on cell proliferation in MCF-7 estrogen responsive cells were not significantly different from low dose estradiol. Low dose Promestriene (2-10pg/ml), unlike comparable doses of estradiol, did not stimulate GREB1 expression in MCF-7 cells. In contrast, T-47D and BT-474 cell lines proliferated and increased GREB- expression in response Promestriene or Fulvestrant treatments.

Conclusions: The potential estrogen-like properties of Promestriene to stimulate the growth of estrogen receptor responsive breast cancer cell lines, especially in estrogen-deprived conditions, suggest caution when prescribed for vaginal atrophy in post-menopausal breast cancer survivors on an aromatase inhibitor. Its ability to activate growth and gene expression in ER- breast cancer cells warrants further study.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-07.
Abstract P5-05-09: Extract ERr731® does not mimic estrogen stimulated growth of MNU-induced breast cancer in ovariectomized Sprague-Dawley rats
Authors: Vollmer G , Möller FJ , Zierau O , Macejova D
Published: August 2014

Background: ERr731® (trade names Phyto-Strol® or femi-loges®), an extract isolated from the roots of the Siberian rhubarb (Rheum rhaponticum), is used as an alternative treatment option for menopausal complaints. In addition, it is regularly used for the treatment of oligomenorrhoea or amenorrhea. The efficacy of the extract on treatment of menopausal complaints was shown in clinical trials and follow-up studies. Although clinical trials did not show changes of mammographic density, we addressed the question whether ERr731 is capable to stimulate experimental breast cancer in an estrogen dependent manner.

Objective: The aim of the study was to assess whether or not the special extract ERr731® in a dosage relevant to human applications is capable to stimulate the growth of methyl-nitroso-urea (MNU) induced experimental breast cancer in ovariectomized Sprague-Dawley (SD) rats.

Experimentals: 32 female SD rats were obtained at the age of postnatal day (PND) 40. On PND 49 and 56 all animals received an intra peritoneal MNU injection of 50 mg/kg body weight (BW). 28 days after the second injection 24 animals were ovariectomized (OvEx) and randomly allocated to 3 experimental groups, 8 animals were SHAM operated and served as positive control. SHAM operated animals (first positive control) and one group of OvEX animals (negative control) were fed a (phyto)estrogen-free diet. A second positive control group of OvEx animals received the phytoestrogen-free diet supplemented with 10 mg/kg diet of estradiol-benzoate. To mimic human exposure, ERr731®, in a dose of 0.067 mg/kg BW/d was chosen which is equivalent to 2.5 mg of the extract/kg chow. The experiment was terminated 25 weeks after MNU injection. Tumor-specific parameters, as well as uterine wet weights (UWW) were assessed.

Results: Ovariectomy reduced the UWW > 6fold. Estradiol-benzoate restored UWW almost completely, whereas ERr731® did not lead to an increase in UWW. Tumor incidence in estrogenized animals was 62.5% (5/8 animals) in the SHAM group and 50% (4/8 animals) in the estradiol-benzoate treated group. No tumors were detected in OvEx negative controls and OvEx animals supplemented with the human relevant dose of ERr731®. The total numbers of tumors were 17 in the group of SHAM operated animals and 5 in the estradiol-benzoate treatment group.

Conclusions: The human dose of ERr731® did neither exhibit estrogenic properties in the uterotrophic assay nor did it stimulate growth of MNU-induced breast cancers in OvEx SD rats, whereas estradiol supplementation at a low dose as well as estradiol levels in animals with a regular estrus cycle significantly promoted tumor growth. In this experimental model the used human dosage of ERr731® therefore appears safe regarding estrogen dependent growth stimulation of experimental MNU breast cancers.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-09.
Abstract P5-07-07: Prognostic value of mutant TP53 in basal breast cancer
Authors: Park JY , Choi JE , Lee JH , Son GT , Kang SH , Bae YK , Lee SJ
Published: August 2014

Backgrounds; p53 is a tumor suppressor gene that plays an important role in cell cycle control and apoptosis. In the breast cancer, mutant tumor protein (TP53) is expressed in approximately 30% and patients with mutant TP53 often tend to have poor response to chemotherapy and poor prognosis than those with normal TP53. But, according to a recent study, TP53 inactivation could cause to significant DNA damage and to eventual cell death by mitotic catastrophe. We investigated the expression frequency and prognostic value of mutant TP53 using tissue microarrays of 898 invasive breast cancers.

Patients and methods; From January 1995 to December 2005 at Yeungnam university hospital, patients who diagnosed with the primary invasive breast cancer and received operation were included in this study. Patients with bilateral breast cancer or distant metastasis at the time of diagnosis were excluded. According to the immunohistochemical results of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 (HER2), Ki67, epidermal growth factor (EGFR) and cytokeratin (CK) 5/6, we classified patients into 6 subgroups, luminal A, luminal B1, B2, HER2-enriched, normal breast-like (triple negative nonbasal) and basal-like breast cancers. Immunohistochemical staining for TP53 was performed and we defined more than 10% stain of tumor cell as mutant TP53-positive. Distribution and prognostic significance of mutant TP53 in each subgroup was investigated.

Results; In 898 invasive breast cancers, mutant TP53 was identified in 33.5% (301/898). Each expression frequency of mutant TP53 was 10.9% (42/385) in luminal A, 32.1% (45/140) in luminal B1, 50.0% (34/68) in luminal B2, 63.7% (72/113) in HER2-enriched, 54.7% (35/64) in normal breast-like and 57.0% (73/128) in basal-like subtype, respectively. In whole breast cancer patients, patients with mutant TP53 tended to have poor overall survival (OS) and disease free survival (DFS). However, there was no statistical significance (p = 0.187 and p = 0.651). But, in 128 patients with basal-like breast cancer, mutant TP53 showed good prognosis in both OS and DFS (p = 0.003 and p = 0.021). In basal-like breast cancer, the expression of mutant TP53 had no association with other clinicopathologic factors such as tumor size, lymph node metastasis, histological grade, lymphovascular invasion etc. and 98.4% (126/128) patients received adjuvant chemotherapy. In multivariate anaylsis, expression of mutant TP53 was an independent prognostic factor for OS and DFS in basal-like breast cancers (p = 0.008 and p = 0.012).

Conclusions: This study showed that basal-like breast cancer with mutant TP53 has a good outcome in both OS and DFS. Further studies are needed to identify the action mechanism of mutant TP53.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-07.
Abstract P5-07-09: An aberrant spliced transcript of focal adhesion kinase expressed in human breast cancer
Authors: Yao L , Peng W , Li S , Shao Z
Published: August 2014

Background: Focal adhesion kinase is a tyrosine kinase that plays a critical role at focal adhesion sites in cell mobility and migration. The over-expression and mutation of FAK, which promotes tumor cell invasion and survival, frequently occurs in tumors, allowing FAK to be recognized as a cancer-relevant gene and a potential target for cancer treatment. The vast majority of evidence has revealed that the deregulation of alternative splicing processes often occurs in cancer-associated genes and leads to tumorigenesis.

Results: From 102 breast cancer tissue samples, we found six samples contained the 26 exon deletion FAK associated with a mutation at the 3192 bp site(CDS), but this 26 exon deletion FAK was not observed in the samples derived from 101 normal tissues. Interestingly, the 26 exon deletion FAK, which possesses the same kinase activity and biological function as wild-type FAK, is resistant to caspase-mediated cleavage and prevents breast cancer cells from apoptosis, promoting breast cancer cells survival.

Conclusion: Therefore, our study revealed that the aberrant splicing of the FAK gene may promote tumorigenesis, providing a new method for diagnosing breast cancer and a potentially important target for breast cancer treatment.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-09.
Abstract P5-08-01: Systematic interrogation of resistance to HER2-directed therapy identifies a survival pathway activated by PRKACA and PIM1
Authors: Moody SE , Schinzel AC , Singh S , Izzo F , Strickland MR , Luo LY , Thomas SR , Boehm JS , Kim SY , Wang ZC , Hahn WC
Published: August 2014

Amplification and/or overexpression of the receptor tyrosine kinase HER2 occurs in 20-25% of breast cancers, and is associated with poor prognosis. Targeting of HER2 with drugs such as trastuzumab, lapatinib, or pertuzumab has led to clinical benefit in patients with both metastatic and early-stage HER2-amplified breast cancer. However, resistance and disease progression always occurs in patients with metastatic disease, and many patients with early-stage breast cancer experience recurrences despite adjuvant anti-HER2 therapy. As such, understanding the mechanisms of resistance to anti-HER2 therapy has important clinical implications.

Recent studies have identified mutations in PIK3CA, the gene encoding the catalytic subunit of Phosphatidylinositol 3 kinase (PI3K), as one mechanism of resistance to trastuzumab. However, such mutations are present in only a fraction of trastuzumab-resistant breast cancers. We therefore sought to uncover novel mechanisms of resistance to anti-HER2 therapy through an unbiased screen for kinases and kinase-related molecules that are able to rescue HER2-amplified breast cancer cells from HER2 inhibition.

We utilized a library of nearly 600 lentivirally-delivered open reading frames (ORFs) to constitutively express the coding sequence of each molecule individually in HER2-amplified BT474 breast cancer cells in arrayed high-throughput format. We conducted two parallel screens for the ability of each of these molecules to rescue cells from anti-HER2 therapy: one in which we treated the cells with a lapatinib-like drug that inhibits the kinase activity of HER2 and EGFR, and one in which we lentivirally delivered a short hairpin RNA that suppresses expression of HER2.

We identified those ORFs that restored viability of BT474 cells to greater than two standard deviations above the median of all ORFs in each screen. Multiple members of the MAPK and PI3K signaling pathways scored in both screens, serving to validate the approach. In addition, the survival kinases PIM1 and PRKACA scored robustly. Mechanistic studies suggest that these kinases may confer resistance by restoring the phosphorylation of, and thereby inactivating, the pro-apoptotic protein BAD. Consistent with this finding, overexpression of Bcl-xl, which is inhibited by BAD, also conferred resistance to lapatinib in HER2-amplified breast cancer cells. Furthermore, pharmacological blockade of Bcl-xl and Bcl-2 with ABT-263 enhanced lapatinib-induced killing of HER2-amplified breast cancer cells in vitro, and partially abrogated the rescue conferred by both PRKACA and PIM1. These findings suggest that combined inhibition of HER2 and the anti-apoptotic molecules Bcl-xl and Bcl-2 could enhance tumor cell eradication and prevent or delay the emergence of resistant disease.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-01.
Abstract P5-08-14: Deregulated histone and cell cycle pathways are associated with anthracycline resistance in breast cancer
Authors: Braunstein M , Liao L , Lyttle N , Taylor KJ , Krzyzanowski P , Kalatskaya I , Stein L , Marcellus R , Al-Awar R , Bartlett JMS , Spears M
Published: August 2014

Drug resistance in breast cancer is the major obstacle to a successful outcome following chemotherapy treatment. While a well-recognized mechanism of resistance involves upregulation of multidrug resistance (MDR) genes, the complexity and hierarchy of other non-MDR driven pathways are still largely unknown. The aim of this study was to identify pathways contributing to anthracycline resistance using isogenic drug resistant breast cancer cell lines. We generated MDA-MB-231, MCF7, SKBR3 and ZR-75-1 epirubicin-resistant breast cancer cell lines, all of which were cross-resistant to doxorubicin and SN-38; only SKBR3 cell line was also resistant to taxanes. Epirubicin-resistant cells were morphologically different from native cells, and had alterations in apoptosis and cell cycle profile. Using gene expression and small inhibitor analyses we identified deregulation of histone H2A and H2B genes in all four cell lines. These genes contribute to several biological pathways, which include cell cycle, chromosomal maintenance, epigenetics, RNA polymerase and mitochondrial transcription, as well as post-translational protein modifications. Importantly, histone deacetylase and cell cycle/DNA damage small molecule inhibitors reversed resistance and were cytotoxic for all four epirubicin-resistant cell lines confirming that histone and cell cycle pathways are associated with epirubicin resistance. Our study has established a model system for investigating drug resistance in all four breast cancer subtypes and revealed key pathways that contribute to the molecular mechanisms of anthracycline resistance.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-14.
Abstract P5-11-06: Combination of insulin-like growth factor 1 receptor and PI3K/mTOR inhibition in triple-negative breast cancers
Authors: Liu T , Taliaferro-Smith LD , O'Regan R
Published: August 2014

Triple-negative (TN) breast cancers account for 15%-20% of all breast cancers with limited treatment options and poor prognosis. The poor outcomes seen with TN breast cancers (TNBCs) are in part due to a lack of viable therapeutic targets. Overexpression of insulin-like growth factor 1 receptors results in mammary tumorigenesis and is common in breast carcinomas. However, the use of IGF1R inhibitor alone has produced limited effects on TNBC cells inhibition. NVP-BEZ235 is a potent PI3K/mTOR dual inhibitor and has been shown to be effective in TNBC cell lines, especially for the mesenchymal-like and luminal-androgen receptor subtypes. Unlike rapamycin, which produces a feedback activation of Akt, NVP-BEZ235 alone successfully blocks the Akt activation and effectively inhibits the cells proliferation. Methods: In this study, we examined the effects of a selective IGF1R small molecule inhibitor picropodophyllin (PPP) in combination with NVP-BEZ235 on human MDA-MB-231, MDA-MB-468, HCC1806 and BT549 TNBC cell lines. SRB cell survival assays were performed following PPP or NVP-BEZ235 treatments alone and PPP in combination with NVP-BEZ235. Western blotting was used to detect expression and phosphorylation of down-stream signaling proteins and fluorescence-activated cell sorting (FACS) analysis to assess effects on apoptosis following treatments. Results: The combined IGF1R inhibition with PPP and PI3K/mTOR inhibition with NVP-BEZ235 resulted in significantly greater cytotoxicity than either PPP or NVP-BEZ235 alone in all four cell lines compared to single agent treatments. Notably, this difference was more pronounced at lower concentrations of both drugs (between 7.8 nM-62.5 nM). The combination of PPP and NVP-BEZ235 inhibitors also significantly increased apoptosis via induction of cleaved caspase-3 and suppressed the PI3K/Akt and MEK/ERK signaling cascades in these TNBC cell lines. Conclusion: These results suggest that the combined inhibition of IGF1R and PI3K/mTOR may be a more effective strategy than single inhibition for TNBC treatments and warrant further investigation in in vivo animal studies.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-06.
Abstract P5-13-14: Breast cancer in very young patient is a more aggressive entity independent from breast cancer subtype
Authors: Martínez MT , Peña-Chilet M , Perez-Fidalgo JA , Bosch A , Alonso E , Ferrer J , Burgues O , Bermejo B , Lluch A , Ribas G
Published: August 2014

BACKGROUND AND OBJECTIVES: Breast cancer in very young patients (< 35 years) (BCVY) is an uncommon disease and when it occurs it usually has aggressive biological characteristics. Whether this effect is due to an overrepresentation of aggressive breast cancer subtypes in younger patients or not remains an issue of controversial. The objective of this study was to identify potential differences in the molecular and clinical features of breast carcinomas from patients < = 35 years versus a cohort of older counterparts previously matched by breast cancer subtype.

METHODS: We performed a retrospective analysis of a prospectively maintained database that included 424 patients diagnosed with an invasive breast carcinoma from 1995 to 2012 at Hospital Clinico of Valencia. We selected 89 patients separated in two groups, the study group with very young women ≤ 35 years old and with no BRCA mutation or unknown and a second group with women older than 50 years. Data related to clinical and pathological features from both groups such as tumor size, nodal status, histological grade, Ki 67 labeling oestrogen and progesterone receptor and HER2 overexpression were obtained from medical records and we used the statistic model of chi-squared to compare the two groups.

RESULTS: Of the 89 patients, 43 patients ≤ 35 year were included in the study group (median age 31.4 years, standard deviation (SD): 3.82) and 46 patients >50 years were included in the control group (median age 66.4 years, SD: 10.00). Ductal carcinoma was the most common histological subtype in both groups (88.4% of BCVY and 78.3% of the old woman). The majority of tumors were ER and PR positive in both groups, but younger women had a higher proportion of HER2 positive tumors, although the result was not significant. By subtype 57.4% of BCVY presented an immunohistochemical luminal subtype, compared to 71.7% in older patients. Triple negative and HER2 profiles were 11.6% and 34.8% in youngest versus 15.2% and 13.04% in older women respectively. No statistically significant differences were found in terms of breast cancer subtype (p = 0.279)). However, BCVY had a higher pathological grade (56% younger patients had grade IIII vs 26% in oldest, p = 0.002), higher proportion of Ki67 >30% (32.5% of youngest versus 17.4% in older women, p = 0.008), larger tumors (16.3% of BCVY had size tumor > 5 cm, versus 2.2% in controls, p<0.0001) and more frequent nodal involvement (39.5% in young women vs 28.2% in oldest, p = 0.0264).

CONCLUSIONS: Patients diagnosed with breast cancer at ≤ 35 years present more aggressive tumors compared to older patients. These data suggest that BCVY is a distinct entity, further studies to confirm these findings are needed.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-13-14.
Abstract P6-01-01: Diet-induced obesity increases tumor growth and promotes angiogenesis in a murine model of breast cancer
Authors: Vona-Davis L , Coad J , Olfert M
Published: August 2014

Recent epidemiological studies provide strong evidence suggesting obesity is a risk factor in several cancers, including breast cancer. However, the exact molecular mechanisms by which obesity increases the risk of breast cancer are poorly understood. In this study, we evaluated the effect of diet-induced obesity on breast carcinogenesis in the MMTV-PyMT breast cancer model. Four-week old, female mice (n = 40) were randomized to high fat (HFD) or low fat (LFD) diets for 8 weeks. Body weights were obtained weekly and mice were imaged for mammary tumor volumes by 3D ultrasound. Tumor burden was collected during early and late stages, weighed and fixed for immunohistochemical staining. Blood and tissue from the liver, lung, and mesenteric fat were also collected. We also compared the basal VEGF protein expression in skeletal muscle, adipose tissue and mammary tumors from MMTV-PyMT mice and compared these levels against background nontransgenic FVB/N mice. Average tumor volumes and tumor burdens were markedly greater in the mice fed HFD than in LFD and showed an increased number of metastases to the lung by 2-fold. HFD increased mammary cell proliferation and elevated Chemokine (C-C motif) ligand 2 (CCL2) in the blood, tumor beds and adipose tissue. Histopathological analysis showed more tumor-associated macrophages and increased microvessel density with diet-induced obesity. Significantly more crown-like structures were evident in adipose tissue from obese mice at both early and late stage carcinoma. Adipose tissue of MMTV-PyMT mice exhibited greater VEGF protein expression compared to adipose tissue of control FVB mice, and greater VEGF protein compared to tumor VEGF expression. Our findings demonstrate that diet-induced obesity exacerbates breast cancer progression in MMTV-PyMT mice and suggests that high body adiposity caused by diet-induced changes promotes an inflammatory and angiogenic phenotype in breast cancer. NIH P20 RR016440, P30 R032138 and GM103488, and P20 RR016477.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-01-01.
Abstract P6-04-11: Use of synthetic isoprenoids to target protein prenylation and Rho GTPases in breast cancer invasion
Authors: Chen M , Knifley T , Subramanian T , Spielmann HP , O'Connor KL
Published: August 2014

Tumor metastasis causes the majority of cancer deaths. Dysregulation of small GTPases such as K-Ras and Rho family GTPases (RhoA, RhoC, Rac1 and Cdc42) is critical to drive the invasion and metastasis of a variety of cancers including breast carcinomas. In order to fulfill their biological functions, these GTPases must properly localize subcellularly and associate with cellular membrane. The association of these proteins to membranes is regulated by a series of post-translational modifications including either farnesylation or geranylgeranylation of the C-terminal CaaX motif, thus providing the rationale to target the farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) as the potential anti-cancer drugs. However, FTase inhibitors (FTIs) failed in clinic due to alternative prenylation, which causes K-Ras to be geranylgeranylated, and their inability to target geranylgeranylated proteins such as Rho family GTPases. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are synthetic farnesol or geranylgeraniol analogs that act as alternative substrates for FTase or GGTase but do not support normal prenyl function. Here, we test the ability of our lead PFIs anilinogeraniol (AGOH; farnesol analog) and anilinofarnesol (AFOH; geranylgeraniol analog) to impact the migratory and invasive capacities of breast carcinoma cells. We found that AGOH treatment effectively decreased the invasion of MDA-MBA-231 in a two-dimensional invasion assay at 100 mM while it blocked invasive growth in three-dimensional (3D) culture model in as little as 20 μM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase, which ribosylates and inactivates Rho proteins. The results demonstrate that Rho proteins are essential for the invasive growth of MDA-MB-231 cells. To determine how AGOH impacts Rho signaling, we performed RhoA and RhoC activity assays in MDA-MB-231 and MDA-MB-468 cells. We determined that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA/C activation and invasive growth where it inhibited RhoA and RhoC activation completely at 20 μM and blocked the invasive growth of MDA-MB-231 cells at ≤ 5 μM. Interestingly, both AGOH and AFOH had no effect on the growth of MCF10A cells in 3D culture. Collectively, this study demonstrates that our lead PFI compounds dramatically inhibited breast cancer invasion, at least in part, through affecting RhoA and RhoC function; thus, suggesting that targeting the function of Rho prenylation by PFIs will offer a promising mechanism for the treatment of breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-11.
Abstract P6-05-03: Evaluation of predictive markers for clinical outcome after neoadjuvant radiochemotherapy in breast cancer
Authors: Matuschek C , Boelke E , Nestle-Krämling C , Speer V , Prisack H , Gerber PA , Bojar H , Audretsch W , Roth S , Budach W
Published: August 2014

BACKGROUND:

Neoadjuvant radiochemotherapy (NRT-CHX) in locally advanced noninflammatory breast cancer (LABC) is an innovative method in treating patients. Proliferation markers make up the majority of genes included in RNA-based prognostic gene signatures applied for breast cancer patients. In this subgroup analysis long-term clinical outcome data and predictive factors were analyzed.

PATIENTS AND METHODS:

During 1991-1998, a total of 315 LABC patients (cT1-cT4/cN0-N1) were treated with NRT-CHX. Preoperative radiotherapy (RT) consisted of external beam radiation therapy (EBRT) of 50 Gy (5 × 2 Gy/week) to the breast and the supra-/infraclavicular lymph nodes combined with an electron boost in 214 cases afterwards or -in case of breast conservation- a 10-Gy interstitial boost with (192)Ir after loading before EBRT. Chemotherapy was administered prior to RT in 192 patients, and concomitantly in 113; 10 patients received no chemotherapy. The impact of age, tumor grade, nodal status, hormone and growth factor receptor status (ER, PR, EGFR), p53, ki-67, HER2/neu, and bcl-2 on pathological complete response pCR and disease-free survival were examined in uni- and multivariate terms.

RESULTS:

Hormone receptor status, proliferative activity, bcl-2, EGFR-status and clinical tumor size had a significant impact on predicting neoadjuvant therapy success. Age, cN, grading, p53, and HER2/neu status failed to reach a significant correlation to complete remission. All examined immunohistochemical factors with the exception of EGFR, and all clinical factors displayed an univariately significant impact on DFS (disease free survival). Particularly, while HER-2/neu had no predictive value for pCR it displayed the highest impact on DFS after complete response (n = 92), even in a multivariate setting with clinical tumor size and nodal status. Complementary, p53 was the most superior immunhistochemical factor for prognosis after neoadjuvant incomplete remission (n = 223).

CONCLUSION:

Her2/neu is a predictive marker for overall survival independent from the pCR. It has no predictive value for the pCR. P53 is a prognostic marker for patients with incomplete remission. Prospective studies are needed to evaluate their use for decisions to further individualize adjuvant treatment after neoadjuvant radiochemotherapy.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-03.