bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P4-03-02: Mapping breast tissue types by miniature radio-frequency near-field spectroscopy sensor
Authors: Kaufman Z , Paran H , Haas I , Malinger P , Zehavi T , Karni T , Pappo I , Sandbank J , Diment J , Alweiss T
Published: August 2014

Background: The study goal was to test the ability of a miniature flexible RF sensor (Dune Medical Devices), designed to be mounted on the surface of surgical tools, in measuring and mapping the various breast tissue types in terms of electrical properties. This study was part of the first stage in developing a measuring device for use during biopsy procedures.

Methods: One hundred and two (102) patients undergoing lumpectomy, open biopsy or mastectomy in 3 medical centers between January and October 2012 were enrolled in this study. The device was applied to numerous sites of freshly excised specimens. Measurement sites were accurately marked, to ensure registration between device measurement and histology analysis. The dielectric properties of the various tissue types were derived from matching between measurements and histology. The correlation between device readings and tissue type, as well as the ability of the device to differentiate between malignant and non-malignant sites, was assessed.

Results: A total of 4322 measurements from 102 patients were analyzed. The dielectric properties of 10 tissue types in the low RF frequency (4-30MHz) range were measured, showing distinct differences between the various types. The correlation between the RF measurements, as quantified by a score variable, and the tissue types, is demonstrated, with a correlation coefficient of 0.90 (95% CI: 0.89 - 0.91). Differentiation ability between tissue types is also demonstrated using ROC curve analysis, with AUG of 0.96 (95% CI: 0.95 - 0.97), and sensitivity and specificity of 90.2% (95% CI: 84 - 95) and 90.9% (95% CI: 90 - 92), respectively, at feature sizes at or above 0.8 mm in diameter.

Conclusions: First results from a radio-frequency near-field spectroscopy miniature flexible sensor evaluate the dielectric properties of various breast tissue types. The results also show promise in differentiating between various breast tissue types, and specifically for differentiation between cancer and normal tissues. The sensor's dimensions (radius of ∼1mm) and flexible design enable use in lumpectomy procedures (for margin assessment) as well as in minimally invasive devices and breast biopsy.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-03-02.
Abstract P4-03-08: Pathological analysis of intraductal papillary lesions in 3690 patients
Authors: Xu Z , Xing W , Yang Z , Li Q , Gu Y , Zhang Y
Published: August 2014

Background:Papillary lesions of the breast (PL) account for 1 - 2% in breast tumors and 10% in benign tumors. Ductoscopy due to its high rate (60 times) amplification and visualization of lesions, which makes it possible to directly visualize the duct from the orifice of the duct well into the periphery. It could not only find abnormities which other tests could not provide, but also predict histological diagnosis.However, ductoscopy findings and pathological results are not always consistent. Ductoscopy examine is limited by many factors such as complicated anatomical structure of breast and ductal obstruction, so in some cases it may have missed diagnosis. Lesions could be missed due to extrusion and shedding during ductoscopy examine procedure and preparation of pathological section.

Methods:In this study, we retrospectively studied on 4170 papillary lesions diagnosed by ductoscopy in 3690 patients from April 23, 2008 to October 31st, 2012 and compared incidence of each disease. These cases were evaluated by final histopathology(42.2% solitary papillary lesion, 32.5% multiple papillomatosis, 13.0% premalignant or malignant lesions, 6.5% mammary duct or plasma cell mastitis, 5.7% epithelial hyperplasia). The sensitivity of ductoscopy for PL in this study was 87.8%, and specificity was 89.2%.

Results and Discussion:Spontaneous nipple discharge(SND) is the third third most common presenting symptom of breast disease accounting for 3∼8% of breast symptoms. But in our experience, not all of the intraductal lesions would have SND, especially in those cases which localized more distally. Besides, intraductal lesions in premalignant or malignant lesions cases would have more incidence to localize distally than their benign papilloma. Thus, if we only take ductoscopy on patients who suffer SND, we might have some missed diagnosis for those people who did not suffer SND including malignant lesions.At present, although ductoscopy is not routine examination for breast cancer screening, we consider that for people who share a high-risk of developping breast cancer, although she might not have SND, ductoscopy could be a selective screen tool.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-03-08.
Abstract P4-05-07: Molecular profiles of screen detected breast cancers: Final results of Turkish Bahcesehir breast cancer screening project
Authors: Cabioglu N , Ozkan-Gurdal S , Kayhan A , Ozaydin N , Ozcinar B , Aribal E , Ozmen V
Published: August 2014

Objectives: Turkish Bahcesehir Breast Cancer Screening Project is a 10-year organized population based screening program carried out in one of the largest counties of Istanbul, Turkey. The aim of our study is to examine the biological features of screen detected breast cancers detected during the initial 4-year study period as an interim analysis.

Study Design and Methods: Between January 2009 and May 2013, a total of 6298 women with ages 40–69 years were recruited in this prospective study. Two-view mammographies were obtained by 2-year intervals, and classified according to Breast Imaging Reporting and Data System of the American College of Radiology (ACR). Clinicopathological and biological tumor characteristics were analysed for those diagnosed with breast cancer. Tumors were stained for estrogen (ER) and progesterone receptors (PR), HER2-neu and Ki-67 by immunohistochemistry.

Results: A total of 57 breast cancers (%0.9) were detected during the study period. The median age was 50 (40-70). The majority of patients (72%) were stage 0 or 1, whereas 28 patients (49%) were <50 age. Of 57 tumors, 45 (79%) were invasive cancers and 12 (21%) were ductal carcinoma in situ. Forty-eight patients (86%) underwent breast conservation, whereas 37 patients (65%) had sentinel lymph node biopsy only as axillary procedure. Of 45 invasive cancers, 31 (69%) were ductal carcinoma and 8 (18%) were pure lobular cacinoma. Among 45 invasive cancers stained for ER, PR, HER2-neu and Ki67, the majority of them (91%) were ER or PR receptor positive, whereas 11% were HER2-neu positive and 58% had low Ki67 levels (≤15). As molecular subtypes, the majority of them were found to be either luminal A (57%) or luminal B type (34%), whereas other nonluminal HER2 (4%) and triple negative cancers (5%) were less frequently detected.

Conclusions: Our findings suggest that the majority of screen-detected breast cancers exhibit either luminal A or B subtype. However, more aggressive subtypes such as nonluminal HER2-neu or triple negative cancer are less likely to be detected by mammographic screening programs, requiring other preventive strategies.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-07.
Abstract P4-06-05: Transcriptional heterogeneity correlates with chemotherapy sensitivity in triple negative breast cancer and varies by molecular subtype
Authors: Jiang T , Shi W , Ononye S , Han G , Wali V , Pusztai L , Hatzis C
Published: August 2014

Purpose: Different molecular subtypes of breast cancer are believed to have different degrees of molecular heterogeneity. However, the heterogeneity of breast cancer subtypes has not been formally compared. The goal of this analysis was to develop metrics of global transcriptional heterogeneity, compare heterogeneity across breast cancer subtypes and assess whether heterogeneity metrics correlate with sensitivity to chemotherapy in basal-like cancers.

Methods: Affymetrix U133A gene expression data from 836 breast cancers (basal n = 219, HER2-like n = 147, luminal A n = 343, luminal B n = 127) were analyzed. Among the basal-like cancers, 128 received neoadjuvant chemotherapy (pathologic complete response, pCR, n = 44; residual disease, RD, n = 94). Molecular class was assigned by the PAM50 classifier. We used the average pairwise Pearson distance, the sum of eigenvalues of the correlation matrix and multi-dimensional scaling dispersions of the probe set expression values to quantify heterogeneity. Distribution of each metric within the different subtypes was estimated from 500 bootstrapped cohorts of 80 cases in each subtype. We used analysis of variance (ANOVA) to evaluate the significance of the differences in heterogeneity between subtypes.

Results: All three heterogeneity metrics yielded concordant results. Within subtypes, heterogeneity was greatest in basal-like cancers followed by HER2-like, luminal B and luminal A (see figure). Luminal A cancers were distinctly less heterogeneous than the rest. Basal-like cancers with RD were more heterogeneous than those with pCR. The best metric to separate basal tumors with pCR versus RD was the sum of eigenvalues shown below.

Conclusion: Breast cancer molecular subtypes differ in transcriptional heterogeneity. Luminal A cancers have the lowest within group heterogeneity while basal like cancers have the highest. Among the basal-like cancers, those with high chemotherapy sensitivity (pCR) are more homogeneous than those with RD suggesting that transcriptional heterogeneity might be contributing to increased resistance to therapy in this subgroup.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-06-05.
Abstract P4-07-12: miR-206 inhibits cell migration through direct targeting of the actin-binding protein coronin 1C in triple-negative breast cancer
Authors: Wang J , Tsouko E , Jonsson P , Bergh J , Hartman J , Aydogdu E , Williams C
Published: August 2014

Patients with triple-negative breast cancer (TNBC) have an overall poor prognosis, which is primarily due to a high metastatic capacity of these tumors. Novel therapeutic approaches to target the signaling pathways that promote metastasis are desirable, in order to improve the outcome of TNBC patients. A loss of function of a microRNA, miR-206, is related to increased metastasis potential in breast cancers but the mechanism remains to be elucidated. In this study, we show that miR-206 was decreased in TNBC clinical tumor samples and cell lines whereas one of its predicted targets, actin-binding protein CORO1C, was increased. Expression of miR-206 significantly reduced proliferation by inducing a G1-S cell cycle arrest and migration and also repressed CORO1C mRNA and protein levels. We demonstrate that miR-206 interacts with the 3’-untranslated region (3’-UTR) of CORO1C and regulates this gene post-transcriptionally. Further, silencing of CORO1C reduced tumor cell migration and affected the actin skeleton and cell morphology, similar to miR-206 expression, but did not reduce proliferation. Our findings suggest that miR-206 targets CORO1C in TNBC cells, thereby affecting the actin filaments which results in a repressed tumor cell migration. This pathway is a novel mechanism offering a mechanistic basis whereby the metastatic potential of TNBC tumors could be targeted.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-12.
Abstract P4-09-04: Weight loss and lipid-lowering efficacy of a low carbohydrate, calorie restricted diet intervention on postmenopausal breast cancer patients
Authors: Krie AK , Bohlen KN , Demuth H , Ageton C , Norfolk D , Kittelsrud J , Thomes R , Fahrendorf A , Muth J , Rojas-Espaillat LA , Davies GE , Hu Y
Published: August 2014

Background: Obesity and weight gain are associated with higher rates of breast cancer recurrence and increased mortality. Cardiovascular disease is a leading cause of death in breast cancer survivors. Control of body weight and cardiovascular risk factors are key aspects in breast cancer survivorship. The purpose of our study was to see if a low carbohydrate, calorie restricted dietary intervention could be implemented in a group of obese postmenopausal breast cancer survivors to achieve weight loss and improved lipid profiles.

Methods: Twenty-four postmenopausal estrogen receptor positive breast cancer patients were enrolled on a low carbohydrate, calorie restricted dietary intervention. The dietary intervention utilizes 0.5g protein/pound (actual body weight), less than 40 grams of carbohydrates, and 800-1200 calories per day. Body weight, body fat percentage, blood triglyceride level, and total cholesterol level were measured and monitored, during the dietary intervention.

Results: The average weight loss spanned 23.8 weeks (varying from 5 to 59 weeks). When compared with the mean body weight prior to the dietary intervention, the mean body weight was reduced by 19.9% (equivalent to 43.7 pounds). The mean body fat percentage was reduced by 6.86%. In addition, the dietary intervention significantly decreased the patients’ blood triglyceride level by 33.8% (p = 0.00013). The mean total cholesterol level was decreased by 9.5% with the dietary intervention (p = 0.00498).

Conclusion: This study demonstrated that a low carbohydrate, calorie restricted diet resulted in significant weight loss with improved lipid profiles. This study suggests that this dietary intervention is feasible in postmenopausal breast cancer survivors and a potential strategy for cardiovascular risk reduction.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-09-04.
Abstract P4-12-18: BOLERO-3: Quality-of-life maintained in patients with metastatic breast cancer treated with everolimus plus trastuzumab plus vinorelbine
Authors: Isaacs C , Ozguroglu M , Jerusalem G , Xu B , Láng I , O'Regan R , White M , Fasolo A , Litton J , Toi M , Shen K , Andre F , Vuylsteke P , Zhang Y , Zhang J , Taran T , Wilks S
Published: August 2014

Background: Activation of the PI3K/mTOR pathway is implicated in resistance to trastuzumab. Accordingly, the BOLERO-3 study evaluated the efficacy of adding everolimus (EVE), an mTOR inhibitor, to vinorelbine and trastuzumab. At the final progression-free survival (PFS) analysis, EVE significantly improved PFS vs PBO (hazard ratio [HR] = 0.78; log-rank P = .0067) but EVE-treated patients had higher rate of grade 3/4 toxicity. To further qualify the benefit:risk of adding EVE to trastuzumab-based therapy, per-protocol, patient-reported, health-related quality-of-life (HRQoL) data were analyzed.

Methods: BOLERO-3 is a randomized phase 3, double-blind, placebo-controlled, international multicenter trial. Taxane-pretreated patients (N = 569) with trastuzumab-resistant, HER2+, advanced breast cancer were randomized (1:1) to treatment with EVE or placebo (PBO) plus vinorelbine and trastuzumab. The European Organisation for Research and Treatment of Cancer (EORTC) quality-of-life questionnaire C30 (QLQ-C30) (including the breast cancer-specific BR23 module) was administered at baseline and every 6 weeks thereafter until progression. The QLQ-C30 consists of 30 items combined into 15 subscales, including Global Health Status and functional subscales, where higher scores (range, 0 to 100) indicate better HRQoL. Time to definitive deterioration (TTD) based on a 10% decrease from baseline for GHS and for the physical, emotional, and social function subscales was determined using the Kaplan-Meier method. Treatment arms were compared using a 2-sided log-rank test stratified by prior use of lapatinib.

Results: Overall, there was no significant difference in median TDD of HRQoL between treatment arms. The median TTD in global health status score was 8.3 months for EVE (95% confidence interval [CI], 6.9-11.5) vs 7.3 months for PBO (95% CI, 5.6-10.4; P = .8386). The median TTD in the physical, emotional, and social function subscale scores showed no significant difference between arms. For example, median TTD in the physical function subscale score was 12.0 months (95% CI, 8.3-14.1) for EVE vs 12.5 months (95% CI, 8.3-20.9) for PBO (P = .4251), and median TTD in the emotional function subscale score was 15.2 months (95% CI, 9.2-17.3) for EVE vs 12.5 months (95% CI, 9.7-16.4) for PBO (P = .8140).

Conclusions: These analyses demonstrate that, despite increased frequency of adverse events observed with the addition of EVE to the standard treatment of vinorelbine and trastuzumab, overall and functional HRQoL scores were not negatively impacted in patients with trastuzumab-resistant, HER2+, advanced breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-18.
Abstract P4-12-19: BOLERO-3: Everolimus plus trastuzumab and vinorelbine in Asian patients with HER2-positive metastatic breast cancer
Authors: Toi M , Masuda N , Andre F , Ishiguro H , Fasolo A , Xu B , Jerusalem G , Shen K , Wilks S , O'Regan R , Isaacs C , Zhang Y , Taran T , Yap Y-S
Published: August 2014

Background: Resistance to trastuzumab may occur through activation of the PI3K/Akt/mTOR signaling pathway, the inhibition of which may restore trastuzumab sensitivity. BOLERO-3, a phase 3, double-blind, international trial involving 569 patients with trastuzumab-resistant, HER2-positive (HER2+), advanced breast cancer pretreated with a taxane, recently reported that adding everolimus (EVE; an mTOR inhibitor) to vinorelbine and trastuzumab significantly improved progression-free survival (PFS) vs placebo (PBO) plus vinorelbine and trastuzumab (hazard ratio [HR] = 0.78; log-rank P = .0067). As EVE pharmacokinetics and, therefore, clinical effects may be different in Asian patients, we analyzed the efficacy and safety data from BOLERO-3 for the Asian population.

Methods: Eligible women with trastuzumab-resistant, HER2+, advanced breast cancer who received prior taxane therapy were randomized (1:1) to EVE (5 mg/day) or matching PBO in combination with weekly vinorelbine (25 mg/m2) and trastuzumab (2 mg/kg after 4-mg/kg loading dose). The primary endpoint was PFS by investigator. Secondary endpoints included safety.

Results: Among 569 patients enrolled in this study, 166 (29%) patients were Asian; 88 and 78 were assigned to EVE or PBO arms, respectively. In this subpopulation, adding EVE to vinorelbine and trastuzumab prolonged median PFS compared with the PBO arm (8.3 vs 6.8 months, respectively; HR = 0.83; 95% confidence interval, 0.59 - 1.18). In general, the incidence of all grade adverse events was similar for Asian versus non-Asian patients in the EVE arm (stomatitis, 71% vs 59%; pneumonitis, 7% vs 5%; and infections, 58% vs 70%) and the PBO arm (stomatitis, 31% vs 26%; pneumonitis, 1% vs 4%; and infections, 48% vs 49%). Serious adverse events had a low incidence and included febrile neutropenia (9.1%), neutropenia (2.3%), stomatitis (2.3%), anemia (2.3%), and cataract (2.3%) as the most common among Asian patients in the EVE arm. The incidence of serious pneumonitis was low: Asian (1.1%) versus non-Asian patients (0%) in the EVE arm and 0% versus 1.5%, respectively, in the PBO arm.

Conclusions: Asian patients in the BOLERO-3 trial treated with EVE plus vinorelbine and trastuzumab showed PFS benefits similar to the overall population and had a comparable manageable safety profile. Thus, EVE in combination with vinorelbine and trastuzumab may be considered as a new therapeutic option for Asian women with trastuzumab-resistant, HER2+, advanced breast cancer progressing after taxane-based therapies.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-19.
Abstract P4-12-22: Time to relapse (TTR) is a potential discriminator of clinical outcome in HER2-positive breast cancer (BC) patients (pts) progressing on adjuvant trastuzumab (AdjT)
Authors: Di Cosimo S , Serpico D , Porcu L , Fanetti G , Molino L , Torri V , de Braud F
Published: August 2014

Background: Clinical trials for HER2-positive metastatic BC excluded pts with prior AdjT and/or relapsing within 1 year (yr) from the end of AdjT. With this gap in mind, we aimed to address the presentation, the therapeutic options, and the clinical outcome of BC pts after failure to AdjT. Methods: Medical charts of BC pts relapsing on AdjT and admitted at the National Cancer Institute of Milan were reviewed. Fisher's exact test was used for contingency tables, long-rank test for survival distribution; p<0.05 was considered statistically significant. Data were matched with literature. Results: Forty pts were identified in the 2013 first quarter. Median age at relapse was 49 (26-80) yrs; 67.5% of pts had stage III; 93% neo-/adjuvant anthracycline+/-taxane; 55% hormonotherapy. Median TTR was 23.5 (5.2-108.5) months (mos). Relapses occurred during (early) or after (late) AdjT in 12 (30%) and 28 (70%) pts. Median AdjT cycles were 15 (2-18) and 18 (6-19) in pts with early and late relapses. Early and late relapses shared a similar distribution of hormone receptor positive and negative status (35% vs 25%, p = 0.73). Lung (8% vs 36%, p = 0.12), bone (25% vs 36%, p = 0.72) and multiple (8% vs 36%, p = 0.12) metastases were less likely to occur in pts with early than in late relapses; liver was affected by 42% and 29% of early and late relapses (p = 0.48). T was the 1stline tretament in 77.5% of pts. After a median follow-up of 21.3 (0.6-98.6) mos, 29 pts (72.5%) progressed. Median Time To Progression (TTP) was 12.7 (9.0-15.3) mos, with a response rate (RR) of 61%. Pts relapsing during AdjT had a shorter TTP than pts relapsing after AdjT, 9.2 (95%CI:3.9-13.3) vs 15 mos (95%CI:9.1-27.9), p = 0.048. T was the 2ndline treatment in 65.5% of pts; with a RR 51%. The literature review showed that pts with prior AdjT represent 0.9-11.7% of overall study-population in just 5 of 8 registrational trials in HER2-positive metastatic BC. No trials included pts relapsing during AdjT. Compared to pts from clinical practice, pts of the above trials were less likely to have locally-advanced BC at diagnosis and neo-/adj chemotherapy. Conclusions: T was the choice in both 1st and 2ndline treatment of pts relapsing on AdjT. Anti-tumor activity was consistent accross lines and with literature. These data should be interpreted with caution, as the cross-sectional design may have favored the inclusion of pts at good prognosis. Notwithstanding the above, pts with early relapse to AdjT showed a worse outcome as compared to the rest. Further investigation to tailor therapy for these pts is recommended. Patient data collection continues and an update will be presented at the meeting.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-22.
Abstract P5-04-16: Extracellular matrix 1 (ECM1) knockdown alters cellular characteristics, CD44 expression and other metastasis associated genes in breast cancer cells
Authors: Gomez-Contreras PC , Domann FE , Weigel RJ , Lal G
Published: August 2014

Introduction: ECM1 is known to be over-expressed in multiple tumor cell lines and primary tumors, including breast cancers. ECM1 overexpression is also an independent predictor of poor prognosis in primary breast carcinomas. While similar associations have also been reported in other tumors, the mechanisms by which ECM1 affects prognosis have not been elucidated.

Methods: ECM1 was silenced in the Hs578t and MDA-MB231 breast cancer cell lines using siRNA. The cells were evaluated for morphological changes, migration (wound-healing assay, TScratch program), matrigel invasion and cell attachment. Real Time RT-PCR using a 94-geneTumor Metastasis Array (TaqMan), was performed to assess the effects of ECM1 downregulation on in Hs578t cells. Alterations in RNA expression were validated at the protein level using western blots. Differences between groups were evaluated using the student's t-test and p<0.05 was considered statistically significant.

Results: ECM1 silenced cells demonstrated altered morphology with a more elongated appearance to the cells, when compared those transfected with non-targeting siRNA. Reduced cell migration was noted for both silenced Hs578t and MB231 cell lines (both p<0.0001). Matrigel invasion was reduced in the Hs578t cell line only (p<0.002). however, cell attachment was decreased in both cell lines. (p <0.0001 for Hs578t, and < 0.01 for MB231). ECM1 downregulation affected the expression of several metastasis associated genes including CD44, Metalloproteinase-2, fibronectin at the RNA level. CD44 expression was significantly reduced in ECM1-silenced cells by western blot.

Conclusion: ECM1 appears to be involved in maintenance of tumor cell shape, migration, invasion and attachment properties in the cell lines tested, and also affects expression of CD44 protein, a known tumor prognostic factor. ECM1 may be a key molecule affecting tumor progression and metastasis, which is a complex process involving these components. Our findings suggest mechanisms by which ECM1 overexpression may affect tumor prognosis. Further work is needed to evaluate if altering ECM1 expression may be a novel therapeutic option for these tumors.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-16.
Abstract P5-05-10: Estrogenic pathways exist in TNBC and correlate with androgen receptor status
Authors: McNamara KM , Yoda T , Nurani AM , Miki Y , Suzuki T , Nemoto N , Miyashita M , Tamaki K , Ishida T , Ohuchi N , Sasano H
Published: August 2014

Triple negative carcinoma are a difficult to treat subtype of carcinomas due to the fact they do not express any of the markers at which targeted therapy is directed at such as ER, PR or HER2. Traditionally these carcinomas are referred to as estrogen receptor negative, however in reality the assessment of ER expression is limited to the ERa form; evaluation of ERβ expression in TNBC has been, on the whole, overlooked. A limited number of studies have suggested that in a large proportion of TNBC ERβ continues to be expressed. In this study we chose to focus on the ERβ1 subtype, as this has been previously suggested to be associated with a positive prognosis in TNBC. We sought to examine if there was an overlap between androgen receptor, ERβ1 and AKR1C3 (17βHSD5) expression as these have been suggested, at least in the prostate, to form a steroid metabolism pathway via which the inactivation of androgen can generate estrogenic signals. In addition we also assessed the presence of the canonical pathway of estrogen metabolism via intratumoral aromatase to see if this had a potential biological role in breast cancer.

Following appropriate IRB approval a cohort of 78 samples of triple negative invasive ductal carcinoma from Tohoku University Hospital were collected. These samples were stained for ERβ1 and aromatase. ERβ1 samples were classified as positive or negative using a 10% labelling index criteria while aromatase was classified as positive if greater that 50% of carcinoma cells showed immunoreactivity. The expression pattern of both stains was then compared to a historical set of data regarding AR, AKR1C3 (17βHSD5 staining) and Ki67. In this comparison ERβ1 positivity and aromatase positivity was significantly and positively associated with AR positivity (Chi squared test p <0.01). ERβ1 however showed no relationship to either aromatase expression or 17βHSD5 expression. While both stains showed a trend towards lower Ki67 labelling indexes in conjunction with higher immunoreactivity, neither was significant. In a standard least squares model incorporating AR, ERβ1, 17βHSD5 and AROM positivity was the only factor that had an independent significant effect on Ki67 labelling index was that of AR positivity (Model p<0.05, AR effect p<0.01) with, as we have previously shown, AR positivity being associated with lower Ki-67 labelling index.

These results suggest that while estrogenic pathways remain in TNBC, the dominant steroid metabolism pathway is that of the androgen receptor. However, further research is warranted to fully understand the roles of estrogenic pathways in triple negative disease.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-10.
Abstract P5-06-01: The PI3K inhibitor GDC-0941 combines with trastuzumab for superior anti-tumor efficacy in HER2+ breast cancer models
Authors: De P , Sun Y , Carlson J , Friedman L , Dey N , Leyland-Jones B
Published: August 2014

Background: PI3K-AKT-mTOR pathway signaling is important for the oncogenic function of HER2. Activating alterations of this pathway are frequently observed in HER2-enriched breast cancer and generally herald a poor response and resistance to trastuzumab (T). Purpose: Targeting the PI3K-AKT-mTOR pathway is an attractive strategy in HER2+ breast cancer that is refractory to trastuzumab. The hypothesis is that the suppression of this pathway by pan-PI3K inhibitor, GDC-0941 may lead to overcome trastuzumab-resistance. Experimental Design: The antiproliferative and HER2-mediated cellular signaling (pAKT, pP70S6K, pS6RP, p4EBP1 and p-ERK) effects of GDC-0941 alone and in combination with T were evaluated in HER2 amplified T-sensitive (BT474), T-resistant (BT474HR), and HER2 amplified/PIK3CA mutated (HCC1954, UACC893) BT cell lines by MTT assay and Western blots. Clonogenic growth was tested by 3D ON-TOP assay and apoptosis markers were also tested. Athymic mice bearing BT474 and BT474HR xenograft tumors were treated with GDC-0941 and T (alone and in combination). Results: (1) GDC-0941 exhibited in vitro cell killing activity in MTT assay with IC50's ranging from 0.35 μM to 1 μm and potency was augmented by the addition of T, (2) inhibition of phosphorylation of AKT(S473, T308), P70S6K, S6RP, and 4EBP1(T37/46, T70) was observed following GDC-0941 treatment, and the combination of GDC-0941 and T more effectively blocked the PI3K-AKT-mTOR pathway, (3) GDC-0941 treatment increased apoptosis markers (CL-CASPASE3 and annexinV positivity), (4) GDC-0941 dose-dependently blocked 3D-ON-TOP clonogenic growth of HER2+ cells. This effect was potentiated in the presence of T and (5) in vivo, the combination of GDC-0941 and T significantly reduced established tumor growth in both sensitive (82%) and resistant (79%) models with concomitant decrease of different PD markers. Conclusions: Our data suggest that 1) therapeutic targeting of the PI3K-AKT-mTOR signaling should be effective in abrogating resistance to T therapy in HER2+ BT, and 2) targeting both the HER2 and the PI3K signaling pathways is an attractive strategy to enhance the clinical efficacy of T therapy, as well as to prevent or delay the development of resistance.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-06-01.
Abstract P5-07-02: Mutant GATA3 causes precocious lobuloalveolar development in the mouse mammary gland and is associated with more aggressive human breast cancer
Authors: Kenny PA , Chandiramani N , Peterson EA
Published: August 2014

GATA3 is an essential mediator of mammary gland development and is expressed at high levels in Estrogen Receptor positive tumors. This gene has recently emerged as one of the most frequently mutated genes in Estrogen Receptor positive breast cancer. GATA3 mutant tumors tend to retain and express the wild-type GATA3 allele which, together with the established role for GATA3 in maintaining luminal epithelial differentiation, has led to the suggestion that GATA3 is a haploinsufficient tumor suppressor gene in the mammary gland.

We describe a striking contrast between the GATA3 mutation spectrum in breast cancer with that found in a true GATA3 haploinsufficiency syndrome and propose that this is indicative of a strong selective pressure to mutate and retain expression of a substantial portion of the GATA3 coding sequence. We demonstrate that the proteins encoded by GATA3 mutant alleles lack the capacity to bind to canonical GATA sequence motifs and acquire a new recognition specificity governed by the N-terminal zinc finger domain. Transgenic expression of the mutant protein results in a pronounced precocious lobuloalveolar development in 9 week old female mice, suggesting that the mutant gene is not a null allele and can regulate cell proliferation. In addition, we demonstrate that Estrogen Receptor positive GATA3 mutant human breast tumors arise at earlier ages than Estrogen Receptor GATA3 wild-type tumors, which further emphasizes the clinical relevance of these mutations.

Based on the estimated mutation prevalence from the TCGA study, approximately 22,000 new cases of GATA3-mutant invasive breast cancer are diagnosed in the US each year. Collectively, our data indicate that GATA3 mutant tumors may comprise a breast cancer subset with distinct biological and clinical behavior and that a detailed understanding of the novel repertoire of target genes may yield useful clinical targets for this population.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-02.
Abstract P5-07-10: APOBEC3B drives mutagenesis in breast cancer
Authors: Harris RS
Published: August 2014

A typical breast cancer accumulates hundreds to thousands of somatic mutations. Breast cancer mutation spectra are often dominated by C-to-T transition mutations, which occur in a mostly dispersed manner but can also occur in localized clusters called kataegis. Several lines of evidence indicate that the innate immune DNA cytosine deaminase APOBEC3B is the primary cause of these mutations. First, APOBEC3B mRNA is up-regulated in the majority of primary breast tumors and breast cancer cell lines. Second, endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell line extracts. Third, APOBEC3B knockdown causes a corresponding decrease in genomic uracil concentrations, mutation frequencies, and C-to-T mutation levels in breast cancer cell lines. Fourth, the biochemical signature of APOBEC3B is over-represented in breast cancer mutation spectra. Fifth, breast carcinomas that express high levels of APOBEC3B have twice as many mutations as those expressing low levels. Finally, high levels of APOBEC3B associate with more aggressive disease including shorter durations of survival. All of these data are consistent with a model in which APOBEC3B-catalyzed DNA cytosine deamination provides a chronic source of DNA damage and mutagenesis in breast carcinomas that may help to explain how some tumors evolve rapidly and manifest gross heterogeneity.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-10.
Abstract P5-08-02: New target in the resistant mechanism to trastuzumab
Authors: Vu TT , Zhang Q , Tian L , Shackleford T , Kute T , Claret FX
Published: August 2014

Her2 positive (Her2+) breast cancer (BC) accounts for 18-20% of all breast cancer subtypes and is associated with high risk of death. Trastuzumab, the first Food and Drug Administration-approved targeted therapy for BC, represents a key milestone in the personalized treatment of Her2+ metastatic disease (Her2+ MBC). However, the median duration of response is less than a year, due to either primary or acquired resistance to the therapy. In addition, there is currently no conclusive biomarker for the response of patients to trastuzumab. Therefore, understanding the development of resistance to trastuzumab is of our interest. Recent studies have proposed various potential mechanisms leading to the resistance, including p27 rapid degradation. Previously, our study and other research demonstrated that Jab1 degrades p27 in breast cancer. These findings suggest that Jab1 over-expression contributes to trastuzumab resistance by facilitating p27 degradation.

Jab1/CSN5 (c-Jun activation domain-binding protein 1) is over-expressed in 50% of primary and 90% of metastatic breast cancers, while its expression is low or absent in normal adult breast tissues. We previously identified that high expression of Jab1 is associated with shorter progression-free survival in breast cancer patients. In this study, our preliminary data showed that the knockdown of Jab1 sensitizes the breast cancer cells to trastuzumab treatment in a dose-dependent manner. Mechanistically, we found that Jab1 over-expression is significantly correlated with the activation of Akt pathway in Her2+ breast cancer cells and xenograft model. Interestingly, activated Akt, due to PTEN loss or PI3K activating mutation has been widely implicated in the potential mechanism to trastuzumab resistance. Therefore, our results suggest that targeting Jab1 overcomes the resistance to trastuzumab via interfering with PI3K/Akt pathway.

In general, our study identifies Jab1 as a novel contributor to trastuzumab resistance and elucidates its potential mechanisms of actions. The successful completion of the study can potentially be translated to the clinic for the benefit of patients refractory to the therapy. Also our study suggests Jab1 expression level can be used as a predictive marker for trastuzumab treatment.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-02.
Abstract P5-08-04: Overexpression of CXCR-4 confers trastuzumab resistance in human breast cancer cell lines
Authors: Mehta A , Wang-Kolodji GK , Tripathy D
Published: August 2014

Background: Trastuzumab significantly improves survival in patients with HER2 positive breast cancer; however, de novo or acquired resistance ensues in the advanced setting. We have identified differentially expression genes in acquired trastuzumab resistance cell line models following long-term trastuzumab exposure and have demonstrated consistent overexpression of CXCR-4 at the mRNA and protein levels. The interaction between the cognate ligand CXCL-12 and its receptor CXCR-4 is known to promote malignant cell proliferation and angiogenesis via activation of Akt and MAPK pathways. CXCR-4 shows no to low expression in normal breast epithelium while its expression sequentially increases from precancerous lesions to carcinomas. CXCR-4 overexpression in human breast cancer is also associated with a worse outcome in small studies. We sought to explore the role of CXCR-4 expression on trastuzumab drug responsiveness.

Methods: Trastuzumab-resistant cell lines were generated by serial passaging of HER+ cells lines BT-474 and SKBR-3 for 1 year with continual exposure to 200 μg/ml of trastuzumab. For these resistant cell lines, termed BtRT and SkRT, CXCR-4 mRNA was quantified using qRT-PCR, and CXCR-4 protein expression was quantified by Western blot. Stable clones overexpressing CXCR-4 receptor were selected by transfecting CXCR-4 sequence-containing lentivirus into sensitive parental sensitive BT-474 and SKBR3 cells. Expression of CXCR-4 receptor was assessed by Western blot assay and by flow cytometry. MTT cell viability analysis was performed on these transfected cell lines to characterize trastuzumab dose responsiveness with parental cells as control.

Results: Trastuzumab-resistant cell line BtRT demonstrated a 100-fold increase in IC50 to trastuzumab treatment when compared to parental BT-474 cells and also exhibited a 3-fold further increase in CXCR-4 mRNA and a corresponding increase in CXCR-4 protein. CXCR-4-transfected sensitive BT474 cell lines showed a greater than 100 fold increase in the IC50 value on MTT assay after treatment with trastuzumab as compared to the parental cell line. Semi-quantitative analysis by Western-blot confirmed a 5-fold increase in CXCR-4 expression in CXCR-4-transfected cells as compared to parental cells. CXCR-4 expression in HER2+ human breast tumors and responsiveness in patients treated with neoadjuvant trastuzumab-based therapy will be presented.

Conclusion: CXCR-4 overexpression and activation of the CXCR-4/CXCL-12 pathway contributes to trastuzumab resistance in human breast cancer cell lines. CXCR-4 targeting may represent a therapeutic strategy to improve the efficacy of trastuzumab in HER2-positive breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-04.
Abstract P5-08-10: Upregulation of microRNA-181a promotes metastasis of breast cancer cells following chemotherapy
Authors: Niu J , Shi Y , Wu Z-H
Published: August 2014

Recent studies indicated that genotoxic treatment may promote cancer cell invasion. Moreover, resistance to chemotherapy highly correlates with high metastatic potential in breast cancer patients. Nevertheless, the molecular mechanisms linking chemotherapeutic resistance to metastasis remain poorly understood. Here, we show that microRNA-181a (miR-181a) plays an important role in promoting chemodrug-induced breast cancer metastasis. Treatment with genotoxic drugs increases miR-181a expression in triple-negative breast cancer cells, which is dependent on NF-kB activation by DNA damaging agents. Genotoxic NF-κB activation upregulates IL-6 expression and secretion in breast cancer cells, which leads to STAT3 (signal transducer and activator of transcription 3) phosphorylation and activation. Activated STAT3 translocates onto miR-181a gene promoter region and enhances its transcription in response to genotoxic treatments. Interestingly, chemodrug treatment also induces epigenetic modifications, such as increased histone H3 Ser28 phosphorylation and decreased H3 Lys27 trimethylation, at miR-181a promoter region, which are also required for miR-181a up-regulation. In addition, we found overexpression of miR-181a in breast cancer cells significantly enhanced cell invasion and metastasis, while inhibiting miR-181a reduced cell aggressiveness upon genotoxic exposure. We further show that miR-181a directly suppresses the expression of ataxia telangiectasia mutated (ATM) in breast cancer cells, which plays a pivotal role in DNA damage response and cancer progression. Therefore, our findings support a critical role of DNA damage-induced miR-181a in promoting breast cancer metastasis following chemotherapeutic treatment. Further exploration of molecular mechanisms regulating miR-181a induction and miR-181a-driven aggressive behaviors of breast cancer cells in response to chemotherapy may reveal novel approaches to reduce metastasis and improve therapeutic response in breast cancers by targeting miRNAs.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-10.
Abstract P5-09-19: Progesterone receptor isoform A as a predictive marker of antiprogestin therapy in breast cancer: A tissue culture method to evaluate drug responsiveness
Authors: May M , Rojas P , Sequeira G , Martinez P , Gonzalez P , Gass H , Lanari C
Published: August 2014

Two thirds of breast cancers express steroid hormone receptors and are susceptible to an endocrine therapy usually aimed to target the estrogen receptors (ER). There is however compelling evidence suggesting that progesterone receptors (PR) participate together with ER, modulating breast cancer growth. Based on our results obtained in an experimental breast cancer model in which antiprogestins induce complete regression of carcinomas with higher levels of PR isoform A (PR-A) than isoform B (PR-B), we propose that antiprogestins such as mifepristone (MFP) might be a possible therapeutic option for patients expressing high levels of PR-A. The goal of our study is to correlate the ratio of PR isoform expression, with the in vitro responsiveness of human breast cancer samples to MFP. Preliminary studies indicated that primary cultures of breast cancer samples overexpressing PR-A were responsive to MFP (10 nM) in vitro. However, since the rate of successful primary cultures was not encouraging (7%), we decided to develop a tissue culture assay to evaluate MFP responsiveness of breast cancer samples in vitro.

Forty five breast cancer samples obtained from the General Pacheco´s Hospital, Buenos Aires, were kept in culture medium at room temperature and in dry ice. The former were cut in slices of 100 μm using a tissue chopper usually used to slice mouse nervous tissue for short incubations, and were incubated on filters in the presence of growth medium (fetal calf serum 10%) with or without MFP (10 nM), for 48 hs at 37º C. The slices were then fixed, paraffin embedded, and processed for Ki-67 staining to evaluate proliferating cells. Frozen samples were processed for Western blotting and the ratio of both PR isoforms was determined in nuclear extracts. In 17/45 (38%) of the samples valuable information was obtained. This was related with the quality (size and amount of viable cells) of the sample. The number of Ki-67 positive cells over the total viable cells was evaluated by a pathologist (M. May) in at least six different control or MFP-treated slices. From nine cases in which the Ki-67 index in control slices doubled the value obtained in MFP-treated slices (p<0.05), 8 were samples in which PR-A was higher than PR-B. MFP increased (p<0.05) the Ki-67 index in one sample with higher levels of PR-B than PR-A. No significant differences were observed in the remaining cases (null, equimolar or higher levels of PR-B than PR-A). We still plan to increase the number of samples in our study. Our data suggest that the tissue culture method developed is much more efficient than the primary cultures to evaluate drug or hormone responsiveness in breast cancer tissue. The results obtained herein support the proposal of PR-A as a predictive marker of antiprogestin therapy in breast cancer patients.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-19.
Abstract P5-11-05: Overexpression of girdin in nucleus indicates poor disease-free survival in ER-positive breast cancer
Authors: Peng W-T , Hu X , Yao L , Jiang Y-Z , Yu K-D , Shao Z-M
Published: August 2014

Background

Girdin is identified as a novel modulator of the PI3K-Akt signaling pathway in regulating neurons integration during neurogenesis process. Recently, several studies demonstrated that Girdin and its phosphorylation type (p-Girdin) were also involved in tumor metastasis and angiogenesis. However, the clinical implications of Girdin and p-Girdin in breast cancer remain unclear. In this study, we detected the expression pattern of p-Girdin (Ser1416) and Girdin in breast cancer to uncover the association between p-Girdin/Girdin status and clinicopathological parameters like estrogen receptor (ER), progestogen receptor (PR), and human epidermal growth factor receptor 2 (Her2), etc.

Methods

This study included 250 patients who were histologically confirmed as invasive ductal breast cancer and underwent mastectomy in the Department of Breast Surgery in Shanghai Cancer Center during 2001 to 2006. We performed immunohistochemistry of p-Girdin/Girdin on tissue microarrays constructed from above specimens.

Results

We observed that Girdin was mainly located in the cytoplasm of mammary epithelial carcinoma cells (195/248, 78.63%) while p-Girdin was mainly located in the nucleus (160/250, 64.00%). However, Girdin/p-Girdin was also weakly expressed in the nucleus/cytoplasm (77/248, 31.05% for Girdin and 193/250, 77.2% for p-Girdin). By the Chi-square test, we found that Girdin in the nucleus (GN) was positively associated with ER status (P = 0.012); Girdin in the cytoplasm (GC) was positively associated with ER (P = 0.004), PR (P = 0.028), and Her2 status (P = 0.004); p-Girdin in the cytoplasm (pGC) was positively associated with PR status (P = 0.001) and negatively associated with tumor size (P = 0.025). However, no significant association was observed between p-Girdin in the nucleus (pGN) and other parameters. Using Kaplan-Meier method, we disclosed a trend that GN indicated poor disease-free survival (DFS) in ER-positive breast cancer (P = 0.075). After adjusted for histological grade and tumor size, multivariate cox regression analysis confirmed this trend (HR = 3.051, 95% CI: 1.137-8.191, P = 0.027). Furthermore, GC was prone to associating with poor DFS, especially in stage II (P = 0.079) and grade III (P = 0.058) patients.

Conclusions

Our work reveals, for the first time, that the location and expression pattern of both Girdin and p-Girdin (Ser1416) in breast cancer, suggesting that p-Girdin/Girdin are associated with ER, PR and Her2 status. In addition, Girdin in the nucleus is associated with poor DFS in ER-positive breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-05.