bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P4-09-02: Omega-3 ethyl esters suppress breast cancer growth by modulating inflammatory signaling
Authors: Chen CH , Quach D , Bowers L , Fabian C , Hursting S , deGraffenried L
Published: August 2014

Introduction: Inflammation has been identified as a key contributor to breast cancer development. Both clinical and preclinical studies confirm a role for inflammatory mediators such as cytokines, interleukins, embedded immune cells and prostaglandins in promoting development of breast cancer. Elevated levels/activity of cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) are correlated with a more aggressive disease. Omega-3 fatty acid (n-3) intake is correlated with an inverse risk for breast cancer development and improvement in prognostic markers. One key target of omega-3 fatty acids is the COX-2 enzyme. We hypothesize that one mechanism by which omega-3 fatty acids suppress breast cancer progression is through inhibition of inflammatory signaling.

Methods: The impact of omega-3 ethyl esters (n-3 EE), a component of some omega-3 supplements, on the viability of MCF-7 breast cancer cells grown in a pro-inflammatory environment was assessed by MTT analysis and on proliferation by cell counting. Supplementation of the growth media with IL-6 (10ng/mL) was used to simulate a pro-inflammatory environment. Changes in expression levels of key components of inflammatory pathways were assessed by Western blot analyses and quantitative PCR. Prostaglandin E2 (PGE2) levels were measured using ELISA assays.

Results: A significant suppression in IL-6-induced proliferation was observed when cells were exposed to physiological concentrations (20 uM) of n-3 EE for 96 hrs. Molecular analyses suggest that the suppression of the NF-kB/COX-2/PGE2 signaling axis was important for mediating this effect. These results are consistent with other studies using specific COX-2 inhibitors.

Conclusions: With their potent anti-inflammatory activity, n-3 EE may prove useful in reducing malignancy of breast cancer and also slow development of new breast cancers. Importantly, they appear to have none of the toxicities associated with pharmaceutical COX inhibitors (NSAIDs). Future studies are planned to incorporate nutraceutical compounds to standard therapy to improve efficacy and reduce associated side effects.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-09-02.
Abstract P4-09-06: No inverse association between breast cancer risk and n-3 unsaturated fatty acids in erythrocytes among Japanese women
Authors: Koriyama C , Kijima Y , Natsugoe S , Akiba S
Published: August 2014

Dietary intake of fish, a major source of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), has been proposed to reduce the cancer risk. In contrast to the animal studies, their protective effects for breast cancer are equivocal in epidemiological studies. In a case-control study, we examined the association between the ratio of DHA (EPA) to aracidonic acid (AA) in erythrocytes and breast cancer risk in Kagoshima, Japan. All subjects, 147 breast cancer cases and 253 controls, were interviewed and information on lifestyles including smoking and alcohol drinking habits, menstrual and reproductive history, family history of cancer, and physical activity, was collected using a questionnaire. Erythrocyte fatty acids were measured by gas-liquid chromatography. According to the results of questionnaire survey, there was no inverse association between the frequency of fish intake and the risk of breast cancer. In pre-menopausal women, there was no significant association between DHA / AA and breast cancer risk (P for trend = 0.953). For post-menopausal women, although the first quartile of DHA / AA showed the lowest breast cancer risk, the risk was the highest in the second quartile and decreased after that. There was no association between EPA / AA and breast cancer risk in both pre- and post-menopausal women. These results suggested no inverse linear association between DHA(EPA) / AA in erythrocytes and breast cancer risk.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-09-06.
Abstract P4-10-01: High dose omega-3 fatty acid supplementation modulates breast tissue biomarkers in post-menopausal women at high risk for development of breast cancer
Authors: Fabian CJ , Kimler BF , Petroff BK , Zalles CM , Metheny T , Nydegger JL , Box JA , Phillips TL , Hidaka BHH , Carlson SE , deGraffenried LA , Hursting SD
Published: August 2014

Background: We conducted a pilot study of high dose omega-3 fatty acid (FA) supplementation in post-menopausal women to determine if risk biomarkers for breast cancer in benign breast tissue sampled by random peri-areolar aspiration (RPFNA) could be favorably modulated and to acquire preliminary data on possible mechanism of action.

Methods: 35 post-menopausal women at increased risk for breast cancer were accrued to a trial of 6-month intervention with 4 g daily of omega-3-acid ethyl esters [1.86 g eicosapentaenoic acid (EPA), 1.5 g docosahexaenoic acid (DHA)]. Subjects had RPFNA performed pre- and post-intervention and specimens evaluated for cytomorphology and proliferation (Ki-67). FA composition was determined in plasma, red blood cells, and RPFNA specimens. Additional specimens were frozen for assessment of hormones, a panel of 11 adipokines and cytokines by Luminex, and gene expression.

Results: 34 subjects completed study with specimens evaluable for change in biomarkers. The ratio of (EPA+DHA):Arachidonic Acid (AA) levels in erythrocyte phospholipid increased significantly by a median of 2.7-fold. Although there was a significant decrease in blood EPA+DHA between discontinuation at 6 months and 2 weeks later when RPFNA was performed, all ratios were above the baseline value (median 1.6-fold). There was favorable but not statistically significant modulation for cytologic evidence of atypia (53% at baseline to 41% at off-study). However, favorable modulation was exhibited for Masood score (medians of 15 to 14; p = 0.014), number of epithelial cells recovered (p = 0.019) and Ki-67 expression (medians of 1.7% to 0.75%, p = 0.036, despite 8 subjects having no Ki-67 expression at baseline). Luminex assay of serum indicated a statistically significant increase (p = 0.003) for adiponectin and decrease (p = 0.016) for TNF-alpha between baseline and off-study. For RPFNA specimens, there was a significant decrease (P = 0.001) in MCP-1 levels adjusted for protein content. By ELISA, serum high molecular weight adiponection increased (p = 0.046) and molar ratio of IGF-1:IGFBP3 decreased (p = 0.006). Note that all analyses were exploratory and without correction for multiple analyses.

Conclusion: Favorable modulation of a variety of blood and tissue risk biomarkers, including cytomorphology and proliferation, along with good tolerability suggests that high dose omega-3 FA esters should be tested further in a placebo-controlled trial.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-10-01.
Abstract P4-11-02: Mammary tumor formation induced by N-methyl-N-nitrosourea (MNU) is accelerated by natural and synthetic progesterone but suppressed by an anti-progesterone CDB4124
Authors: Lee O , Sullivan M , Ivancic D , Shidfar A , Wiehle R , Khan SA
Published: August 2014

Background: CDB4124, anti-progesterone suppresses the development of carcinogen-induced ER+/PR+ mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer. We hypothesize that progesterone (P4) and medroxyprogesterone acetate (MPA) will accelerate mammary carcinogenesis induced by MNU, however CDB4124 will efficiently suppress tumor formation stimulated by progesterone.

Methods: ovary intact female Sprague Dawley rats received a single intraperitoneal injection of 50mg/kg MNU at 4-5 weeks of age. 30mg of CDB4124 and 25mg of P4 or MPA (90 release pellets, Innovative research of America, Inc) were implanted in dorsal area at 3 weeks and 4 weeks after MNU injection, respectively. 10-11 rats were used for each treatment group. Tumor incidence, latency, multiplicity, and burden were recorded weekly. 9 weeks after MNU injection all the mice were euthanized and mammary tumors and glands were fixed in 10% (v/v) neutral buffered formalin. Plasma concentrations of CDB4124 and its metabolite CDB4453 were determined by LC-MS/MS.

Results: The first tumor appeared in the control group at 5 weeks, and in the P4 and MPA treated groups at 6 weeks after MNU injection. 7 weeks after MNU injection, mammary tumor incidence of MPA and P4 treated groups were 80% and 50%, respectively compared to 30% in the control group. 9 weeks after MNU injection all MPA treated mice, 80% of P4 treated mice, and 60% of control mice developed tumors. Tumor incidence, latency, multiplicity, and tumor weight were summarized as mean ± SD in Table 1.

[Table 1] Tumor latency, incidence, multiplicity, and burden in mammary tumorsTreatmentsLatency (days)Incidence(%)MultiplicityBurden (g)Control53.7 ± 12.9602.34.25 ± 7.02P453.6 ± 9.6802.64.11 ± 5.32MPA50.8 ± 7.71002.56.02 ± 4.85P4 + CDB412459.3 ± 11.53621.38 ± 0.61MPA + CDB412454.3 ± 10.7732.84.19 ± 4.39

Tumor latency of CDB4124 treated groups was increased; tumor incidence and burden (g) of CDB4124 treated groups were decreased compared to P4 and MPA treated groups. In particular, tumor incidence and burden of CDB4124 + P4 treated group were significantly lower than those of the control group. Plasma CDB4124 and CDB4453 were 11.6 ±5.88 ng/mL and 3.4±1.68 ng/mL, respectively. Histopathology of tumors and mammary glands and immuno-histochemical evaluations of Ki67, activated caspase-3, CD34, ER, and PR are currently underway.

Conclusions: Our results indicated that natural progesterone promotes MNU- induced mammary tumor formation similar to synthetic progesterone, MPA in rats. Under this tumor permissive environment, CDB4124 provided excellent prevention efficacy, suggesting good potential as breast cancer prevention agent.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-11-02.
Abstract P4-12-06: Small HER2-positive breast carcinomas: Prognostic factors to consider in deciding on adjuvant trastuzumab treatment
Authors: Campiglio M , Sandri M , Sasso M , Bianchi F , Turdo F , Ménard S , Tagliabue E
Published: August 2014

Criteria for the decision to treat small tumors in adjuvant setting remain debatable, especially for HER2-positive tumors, since HER2 itself is associated with poor prognosis. Analysis of a large database including 503 T1 HER2-positive breast carcinomas treated with chemotherapy plus trastuzumab in Italian oncology clinics from 2006 to 2009 and, with 32 months of median follow-up and with 35 (7.0%) relapses, revealed no statistically significant differences in relapse rate among the T1a/T1micro, T1b and T1c groups, suggesting that tumor size is not associated with relapse probability in T1 HER2-positive tumors; the only clinico-pathological factors significantly associated with disease-free survival were nodal involvement (unadjusted HR = 3.4, 95%CI = 1.6-7.3, p = 0.002), loss of hormone receptor expression (HR = 3.1, 95%CI = 1.5-6.2, p = 0.002) and high tumor grade (HR = 2.9, 95%CI = 1.2-6.9, p = 0.020). Multivariate analysis indicated that patients who were both ER-negative and lymph node-positive presented the highest risk of relapse (HR = 5.5, 95%CI = 2.8-10.9, p = <0.001). These results suggest that the clinical decision for adjuvant treatment of small HER2-positive tumors should be based on nodal and hormone status, not on tumor size information. While the most recent National Comprehensive Cancer Network guidelines (version 3.2012) for systemic adjuvant treatment of small HER2-positive breast cancer only suggest considering adjuvant chemotherapy treatment including trastuzumab for T1a cases with axillary node micrometastasis and for all T1b, but require such treatment for all tumors >1 cm, our data identify T1 tumor ER-negativity/axillary lymph node-positivity as the crucial criteria for adjuvant treatment, independent of tumor size categories.

Supported by AIRC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-06.
Abstract P4-12-08: Final efficacy results from a single-arm multicenter phase II study evaluating first-line trastuzumab, bevacizumab, and capecitabine (HAX) therapy for HER2-positive locally recurrent/metastatic breast cancer (LR/mBC)
Authors: Martín M , Makhson A , Gligorov J , Lichinitser M , Lluch A , Semiglazov V , de Ducla S , Mitchell L , Tjulandin S
Published: August 2014

Background: After median follow-up of 8.8 months, the primary efficacy results from the HAX study [Martín et al., Oncologist 2012] demonstrated a 73% overall response rate (ORR) according to RECIST (primary endpoint), median progression-free survival (PFS) of 14.4 months and a safety profile typical of the component agents; overall survival (OS) data were immature at that time. We present final efficacy results.

Methods: Patients with measurable HER2-positive LR/mBC who had received no prior chemotherapy for LR/mBC received bevacizumab 15 mg/kg day 1, trastuzumab 6 mg/kg day 1 (8 mg/kg loading dose in cycle 1), and capecitabine 1000 mg/m2 bid days 1–14, repeated every 3 weeks until progression, unacceptable toxicity, or withdrawal of consent.

Results: At the time of data cut-off, median follow-up was 31.8 months (range 1.2–44.5 months). Median duration of study therapy was 9.0, 9.4, and 10.3 months for bevacizumab, capecitabine, and trastuzumab, respectively. More than 8 cycles were administered in 67%, 65%, and 70%, respectively. Efficacy results in the intent-to-treat population (n = 88) and clinically important subgroups are shown below.

Patient subgroupBest ORR,% (95% CI)PFS events, n (%)Median PFS, months (95% CI)Deaths, n (%)Median OS, months (95% CI)1-year OS rate,% (95% CI)All patients (n = 88)75 (65-84)70 (80)14.2 (10.5-14.9)40 (45)31.8 (26.3-38.2)94 (86-98)ER and/or PgR positive (n = 37)73 (56-86)32 (86)14.1 (8.5-17.0)19 (51)31.8 (24.4-42.6)100 (*-*)ER and PgR negative (n = 51)77 (63-87)38 (75)14.5 (10.5-18.9)21 (41)33.1 (23.6-44.5)90 (77-96)No prior chemotherapy (n = 37)81 (65-92)27 (73)14.9 (10.5-24.8)14 (38)31.8 (26.3-44.4)94 (80-99)Prior chemotherapy (n = 51)71 (56-83)43 (84)13.6 (8.5-14.8)26 (51)30.9 (24.3-42.1)94 (82-98)Taxane pretreated (n = 28)57 (37-76)26 (93)9.5 (5.2-14.0)17 (61)27.4 (21.4-33.2)93 (74-98)Prior anti-HER2 therapy (n = 22)73 (50-89)18 (82)12.1 (5.2-17.0)11 (50)30.9 (24.3-*)95 (72-99)Age >60 years (n = 25)72 (51-88)16 (64)14.2 (10.5-21.7)11 (44)33.2 (22.0-*)100 (*-*)*Not reached

The most common grade ≥3 adverse events were hand-foot syndrome (26% of patients), diarrhea (11%), and hypertension (11%). The majority of deaths (38/40) were from progressive/underlying disease. The cause of the two remaining deaths was described as unknown; one was reported at the primary analysis and the other occurred 19 months after last study drug administration, following progression. Detailed long-term safety results will be presented.

Conclusion: Long-term follow-up results from the HAX study show that this is an active and tolerable first-line triplet therapy for HER2-positive mBC, demonstrating median OS of 31.8 months.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-08.
Abstract P4-13-06: Ligation of dectin-1 on tumor infiltrating dendritic cells promotes breast cancer rejection
Authors: Wu T-C
Published: August 2014

Human breast cancer microenvironment displays features of T helper2 (Th2) immunity which promotes tumor development via IL-13/IL13 receptor pathway. This is driven by breast cancer cell-derived thymic stromal lymphopoietin (TSLP) which, by inducing OX40L expression on DCs, contributes to the Th2 immunity conducive to breast tumor development.

In order to reprogram the inflammatory pro-tumor Th2 (iTh2) into anti-tumor Th1 microenvironment, we tested the impact of targeting the innate receptors on DCs to render them resistant to tumor environment. We show that intratumoral delivery of b-glucan, a natural ligand for dectin-1 expressed on DCs, blocks the generation of iTh2 cells leading to decreased IL-13 in the tumor microenvironment and prevents breast cancer development in vivo. β-glucan inhibits OX40L expression on tumor-associated DCs which is due to a block in STAT6 phosphorylation. β-glucan-treated DCs, when exposed to breast cancer supernatant, secrete higher levels of IL-12p70 and do not expand iTh2 cells thereby enabling the T helper 1 cells secreting IFN-γ. Importantly, β-glucan exposed DCs expand CD8+ T cells that express CD103, a ligand for E-cadherin. These CD8+ T cells, which produce higher IFN-γ, Granzyme A and Granzyme B, accumulate in the tumors leading to enhanced tumor necrosis in vivo. DC reprogramming by β-glucan is dependent upon dectin-1 engagement. The ligation of TLR7/8 (CL075) and TLR3 (polyI:C) show the same effect in Th2 response inhibition but no effect on the functions of CD8+ T cells. Taken together, our data demonstrate that exploiting pattern recognition receptors on tumor-infiltrating DCs enables cancer rejection.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-13-06.
Abstract P4-14-01: A time course study of genomic instability in breast cancer patients receiving neoadjuvant therapy with or without bevacizumab
Authors: Møller EK , Moen Vollan HK , Nord S , von der Lippe Gythfeldt H , Edvardsen H , Silwal-Pandit L , Krohn M , Fleischer T , Schlitchting E , Borgen E , Garred Ø , Fangberget A , Holmen MM , Skjerven H , Lundgren S , Wist E , Naume B , Børresen-Dale A-L , Kristensen VN , Engebraaten O
Published: August 2014

Tumor heterogeneity is an area of intense research, revealing tumors with high complexity consisting of different subclones and infiltrating cells. Identification of subclones that are resistant to therapy may be critical to improve treatment outcome.

The NeoAva study is a randomized phase II, clinical trial of Her2 negative breast cancer patients treated in a neoadjuvant setting with chemotherapy (FEC and taxane) +/- bevacizumab. Core needle biopsies were obtained at screening and after 12 weeks, and the tumor was surgically removed after 25 weeks. DNA copy number changes in the tumors were analyzed using Affymetrix SNP Array 6.0. Allele specific copy number changes were assessed using the Allele-Specific Copy number Analysis of Tumors (ASCAT) algorithm (Van Loo, Norgard et al., PNAS 2010) and allele-specific Piecewise Constant Fitting (asPCF) algorithms (Nilsen, Liestol et al., BMC Genomics 2012). Measures of genomic instability were obtained through the complex arm-wise aberration index (CAAI) that captures local rearrangements (‘firestorms’) (Russnes, Vollan et al., Sci Transl Med 2010).

Changes in copy number aberrations between the three different time points were observed in almost all tumors. Some tumors showed a decrease in tumor percentage and aberrations after just 12 weeks of treatment, where others showed loss of aberrations only at the time of surgery (25 weeks). Most of the tumors that did retain aberrations at all time points during treatment, did not demonstrate any decrease in tumor size. Other profiles indicated subclonal reduction, where some aberrations are kept throughout treatment and others disappear. Many of the tumors shrinking in size showed fewer whole arm aberrations than before treatment, but retained their focal amplicons. Some of the tumor aberrations seem to disappear after 12 weeks, but to reappear after 25 weeks, but with the addition of novel aberration.

Complex rearrangements were identified in 67% of tumors before treatment. The most frequent ‘firestorms’ were found on 20p, 11q and 8p. Some events were persistent through therapy, but the majority changed. An association between complex tumor genomes and patients having progressive disease/non-responders were observed.

These results show the complex structure of a tumor and suggest that heterogeneity will influence the response to treatment. The subclonal patterns of tumors may be of great importance for clinical decision-making, as well as for monitoring treatment efficacy.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-14-01.
Abstract P4-15-02: The PI3K inhibitor GDC-0032 enhances the efficacy of standard of care therapeutics in PI3K alpha mutant breast cancer models
Authors: Sampath D , Nannini MA , Jane G , Hong R , Parsons KL , Belvin M , Friedman LS , Wallin JJ
Published: August 2014

The phosphoinositide 3-kinases (PI3Ks) are lipid kinases that activate the PI3K signaling pathway and play an essential role in regulating breast tumor cell growth, migration, and survival. Activating and transforming mutations in the PIK3CA gene (PI3K alpha) are commonly found in HER2+ breast cancer and ER+ breast cancer. GDC-0032 is a selective, orally bioavailable inhibitor with a Ki of 0.29 nM for PI3K alpha with 30 fold less inhibition of PI3K beta. In addition, GDC-0032 has increased single agent activity against PI3K alpha mutant and HER2 amplified cancer models in vitro and in vivo. The aim of our study was to evaluate the efficacy of GDC-0032 in breast cancer models in combination with standard of care therapeutics including taxanes, endocrine therapies, and HER2 targeted therapies.

We evaluated cell viability for a range of dose concentrations of GDC-0032, as single agent and in combinations, in eleven breast cancer cell lines that harbor mutations in PI3K alpha (K111N, E545K, H1047R) and/or over-expressed HER2. GDC-0032 was combined with taxanes (paclitaxel and docetaxel), endocrine therapies (fulvestrant and tamoxifen) or anti-HER2 agents (trastuzumab, pertuzumab or ado-trastuzumab-emtansine) and synergy quantified using the Chou and Talalay method of Combination Index (C.I.). The combination of GDC-0032 with taxanes, endocrine therapies or anti-HER2 therapies were synergistic in the breast cancer cell lines tested based on C.I. values of less than 1.0. The combination of GDC-0032 with docetaxel in vitro resulted in decreased viability as a result of increased cell death. The in vitro results translated in vivo as GDC-0032 enhanced the anti-tumor activity of docetaxel and paclitaxel in MCF7 (E545K) xenografts that resulted in increased tumor regressions when GDC-0032 was dosed daily or intermittently on the same schedule as the taxane. In addition, the combination of GDC-0032 and fulvestrant or tamoxifen resulted in greater efficacy in the MCF7 xenograft model when compared to either agent alone. Enhanced efficacy was also observed when GDC-0032 was combined with trastuzumab or ado-trastuzumab-emtansine in the HER2+ BT474M1 (K111N) xenograft model. Moreover, the triple combinations of GDC-0032/trastuzumab/pertuzumab or GDC-0032/trastuzumab/docetaxel resulted in durable tumor regressions that were sustained in the HER2+ KPL-4 (H1047R) and BT474M1 xenograft models, respectively. All drug combinations with GDC-0032 were well tolerated in vivo based on minimal changes in body weight. Collectively, our preclinical data demonstrate that GDC-0032 enhances the efficacy of standard of care therapeutics in PI3K alpha mutant breast cancer models and provides a strong rationale for further evaluation in patients.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-02.
Abstract P4-15-07: MicroRNA signature of breast cancer brain metastasis for novel targeted therapies
Authors: Sethi S , Ahmad A , Ali-Fehmi R , Mittal S , Dyson G , Sarkar F
Published: August 2014

Rationale:

MicroRNAs (miRNAs) are recently described novel small non-coding endogenous RNAs which critically regulate cancer progression, invasion and metastasis. Altered expression of miRNAs has been proposed to control the invasive capability of cancer cells. Modulating altered miRNAs is a novel approach for targeted therapy for cancer. Breast cancer is the most common cancer among women in the US. Breast cancer-related mortality and morbidity are primarily due to metastatic disease especially brain metastasis which also impacts patients’ quality of life. Molecular mechanisms of brain metastasis of breast cancer are largely unknown.

Study Objectives:

Identifying molecular miRNA signatures in breast cancers which metastasize to the brain can assist in designing novel targeted therapies to prevent and eliminate brain metastasis.

Design:

RNA was extracted from formalin fixed paraffin embedded tumor tissues from a cohort of breast cancer patients with brain metastasis, and age, stage and follow-up matched breast cancer cases without brain metastasis. miRNA expression profiling was done using EQIXON microarray. Quantitative real-time PCR (qRT-PCR) was used to validate abnormal expression of miRNAs. Data was statistically analyzed using Kruskal-Wallis test to determine the clinical significance of the findings.

Results:

Over 2000 unique miRNA sequences were profiled in each case. The upregulated miRNAs included miR-3927-5p, miR-216b and miR-10b and the down regulated miRNAs were miR-4280, miR-3174 and miR-1244 and miR20b. Validation of the deregulated miRNAs by q RT-PCR showed statistically significant difference in the expression levels in the breast cancers with brain metastasis compared to breast cancers without brain metastasis (Kruskal-Wallis test; p < 0.001).

Conclusions:

A unique set of miRNAs were deregulated in breast cancers with brain metastasis. This panel of altered miRNAs have clinical relevance since they can be silenced by antagomirs (chemically modified anti-miRNA oligonucleotides) to treat and prevent brain metastasis. These molecular microRNA signature-based novel targeted prophylactic and therapeutic strategies could improve patients’ quality of life and overall survival in the era of personalized and precision medicine.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-07.
Abstract P4-15-08: Discovery of a potent inhibitor of CREB-mediated gene transcription that completely suppresses the growth of triple-negative breast cancer cells
Authors: Xiao X , Li B , Xie F , Fan Q , Xue C , Qian D
Published: August 2014

Background: Breast cancers are a heterogeneous group of diseases with distinct and complex mechanisms of pathogenesis. Triple-negative breast cancers (TNBC) form a subgroup of breast cancers with poor prognosis. TNBCs lack the expression of estrogen receptor (ER), progesterone receptor (PR) or HER2 and no targeted therapies exist. The cyclic-AMP (cAMP) response element binding protein (CREB) is a stimulus-induced transcription factor activated by multiple extracellular signals through phosphorylation. The transcription activity of CREB depends on its phosphorylation at Ser133 by mitogen- and stress-activated protein serine/threonine kinases, which are often dysregulated in TNBCs. The phosphorylated CREB (p-CREB) can then bind the mammalian transcription co-activator, CREB-binding protein (CBP), via the kinase-inducible domain (KID) in CREB and KID-interacting (KIX) domain in CBP. This binding event will further recruit other transcriptional machinery to the gene promoter to initiate CREB-dependent gene transcription. CREB is overexpressed in breast cancer tissues compared to normal mammary tissues and the level of expression inversely correlates with disease-free survival. Genetic studies have shown that inhibition of CREB's activity leads to decreased breast cancer cell proliferation. We describe here the discovery of a potent small molecule inhibitor of CREB-mediated gene transcription with in vitro and in vivo activity in TNBCs.

Methods: The small molecule inhibitor (compound 1) was reporter earlier by us (Li and Xiao ChemBioChem 2009). Starting from this lead compound, we designed and synthesized a more potent compound 2. The in vitro antiproliferative activity and apoptosis induction were evaluated in MDA-MB-231 and MDA-MB-468 cells with MTT assays and flow cytometry, respectively. In vivo activity was investigated in a human xenograft model of MDA-MB-468. The CREB target gene expression was investigated by Western blot and qRT-PCR analysis.

Results: Starting from compound 1, which is a low micromolar and cell-permeable inhibitor of CREB-mediated gene transcription, we designed and synthesized compound 2. Compound 2 potently inhibited CREB-mediated gene transcription with IC50 ∼80 nM. In vitro, this compound inhibited proliferation of MDA-MB-231 and MDA-MB-468 cells with concomitant activation of apoptosis and necrosis. In contrast, compound 2 was not toxic to normal human mammary epithelial cells. In vivo, compound 2 completely suppressed the growth of MDA-MB-468 cells at a dose not toxic to the mice.

Conclusion: Compound 2 potently inhibited CREB-mediated gene transcription and TNBC cell growth in vitro and in vivo. These results suggest that CREB is a promising druggable target for TNBCs.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-08.
Abstract P4-16-02: EF24 targets radiation-induced NFkB-dependent stemness in triple negative breast cancer cells
Authors: Ramraj SK , Natarajan M , Aravindan S , Herman TS , Aravindan N
Published: August 2014

Prognosis of patients with late stage BCa still remains poor, mostly due to development of chemoradioresistance followed by tumor recurrence. Cancer stem cells (CSCs), with higher drug efflux capability, and other stem cell-like properties were proposed to be responsible for resistance, relapse and progression of BCa. We have shown that EF24 alleviates radiation (IR)-orchestrated NFkB mediated clonal expansion. Herein, we investigated the potential of EF24 in the regulation of IR-induced NFkB dependent stemness in triple negative breast cancer cells (TNBC). MDA-MB-231 cells exposed to mock-IR or IR (2Gy) with/without EF24 were examined for transcriptional alterations of 93 EMT, CSCs self-renewal, pluoripotentcy maintenance and other stem cell markers. NFkB (p50/p65) overexpression (with or without EF24) and RelA siRNA knockout (with IR) approach were used to delineate the role of IR-induced NFkB and the selective NFkB targeting of EF24 in this setting. Nanog, Sox-2, ABCG-2, N-Cadherin, POU5F1 and Myc expression was examined with immunoblotting. IR profoundly increased the transactivation of 86 stem-cell related molecules in TNBCs that are involved in cell survival. Interestingly, muting IR-induced NFkB attenuated 85 of those genes. Notably, EF24 suppressed identical 85 genes reproducing the inhibitory signature of NFkB muting. Coherently, activating NFkB induced 87 stem-cell related molecules in TNBC and of which 86 genes were completely suppressed with EF24. Alterations in the cellular expression levels of Nanog, Sox-2, ABCG-2, N-Cadherin, POU5F1 and Myc validates the potential of EF24 in mitigating IR-induced stemness in TNBC. Together these data demonstrates, at least in TNBC cells, IR-induced NFkB mediates increased stem-like characteristics and further imply that EF24 may alleviate stemness by selectively targeting IR-induced NFkB.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-02.
Abstract P4-16-03: Effect of doxorubicin and paclitaxel on adipose-derived stem cells and breast cancer cells: Can we incorporate chemotherapy into our reconstructive strategies?
Authors: Tsuji W , Chung CW , McLaughlin MM , Valentin JE , Marra KG , Rubin JP
Published: August 2014

Introduction: Breast surgery often results in physical deformities that significantly diminish patient's quality of life. A natural and aesthetically pleasing breast reconstruction can be achieved through autologous fat grafting and the success of this technique has largely been attributed to the regenerative properties of adipose-derived stem cells (ASCs) within fat graft. However, in the setting of resected breast cancer, the growth stimulating and angiogenic effects of ASCs pose a risk of increasing local recurrence rates. Although the safety of autologous fat grafting in the breast cancer population has yet to be determined, potential recurrence risk may be minimized through incorporating tumor-suppressing elements in the graft. This study aimed to determine if doxorubicin and paclitaxel could be used to inhibit breast cancer cells while maintaining the viability and functionality of ASCs in vitro.

Materials and methods: Human ASCs were isolated from non-diabetic female patients between 35 and 60 years of age (n = 3). BT-474 and MDA-MB-231 were obtained from ATCC. ASCs, ASCs undergoing adipogenic differentiation, and breast cancer cells were each exposed to a range of doxorubicin-HCl (0, 10, 30, 100, 300, 1000, 3000, or 10000nM) or paclitaxel (0, 0.1, 0.3, 1, 3, 10, 30, 100, or 300nM) concentrations. Proliferation, viability, and differentiation capacity were assessed with commercially available CyQuant, MTT, and AdipoRed assay kits, respectively.

Results: Dose-dependent inhibition was observed for doxorubicin in ASCs and both breast cancer cell lines. The IC50 of doxorubicin on ASCs, BT-474, and MDA-MB-231 were 901.3, 656.5, and 333 nM, respectively. Dose-dependent inhibition for paclitaxel was only observed in BT-474 and MDA-MB-231 cells, with IC50 values of 1.809 and 3.477 nM, respectively. ASCs maintained greater than 80% viability over the range of paclitaxel concentrations tested and consequently and IC50 value could not be determined. ASCs differentiation into mature adipocytes was not inhibited by doxorubicin exposure.

Discussion: In vitro cytotoxicity studies demonstrated greater doxorubicin and paclitaxel sensitivity in BT-474 and MDA-MB-231 than in ASCs. Furthermore, the presence of doxorubicin did not inhibit ASC differentiation into mature adipocytes. These findings suggest that incorporating chemotherapeutic drugs in fat grafts for breast reconstruction following primary breast cancer surgery may be a viable option for decreasing the risk of cancer recurrence.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-03.
Abstract P4-16-06: Histone deacetylases inhibitor SAHA enhances anti-tumor effects of poly (ADP-ribose) polymerase (PARP) inhibitor olaparib in breast cancer cells
Authors: Min A , Im S-A , Kim S , Song S-H , Lee K-H , Han S-W , Oh D-Y , Kim T-Y , Bang Y-J
Published: August 2014

Background: The poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib, has been found to have a therapeutic potential for treating cancers that have an impaired DNA repair ability. However, some cancer presents acquired resistance to PARP inhibitor or platinum. Histone deacetylases (HDACs) are important to enable functional homologous recombination (HR) by regulating the expression of HR-related genes and promoting the accurate assembly of HR-directed subnuclear foci. Thus, HDAC inhibitors have emerged recently as a class of therapeutic agents for the treatment of cancer by inhibiting DNA repair. For this mechanism, HDAC inhibition would enhance the anti-tumor effect of PARP inhibitor in cancer cells by blocking DNA repair pathway.

Materials and Methods: We determined whether SAHA, a HDAC inhibitor could enhance the growth inhibition of olaparib on breast cancer cell lines using MTT assay. We examined whether exposure to SAHA affects the expression level of genes involved in HR. The accumulation of DNA double strand breaks (DSBs) induced by combination treatment was accessed by the comet assay. Cell cycle analysis and molecular changes induced by combination of olaparib plus SAHA were also performed. These in vitro data were validated in the in vivo xenograft model as well.

Results: Triple-negative breast cancer cell lines showed heterogeneous response to dual inhibition of PARP and HDACs. SAHA enhanced olaparib-induced cell death of MDA-MB-157 and HCC1143 but not of HCC70 and MDA-MB-468. Combination of olaparib plus SAHA caused a greater decrease of pAKT, pERK, and pSTAT3 in MDA-MB-157 and HCC1143 cells compared with monotherapy either olaparib or SAHA. Furthermore, inhibition of PARP increased the accumulation of DNA DSBs induced by SAHA in these two cell lines, MDA-MB-157 and HCC1143. There was no change in proliferative pathway activation in HCC70 and MDA-MB-468 cells with combination of olaparib plus SAHA. Our findings showed that triple-negative breast cancer cells are differentially effective to combination of SAHA plus olaparib which increased levels of unrepaired DNA DSBs.

Conclusion: HDAC inhibitor SAHA enhances growth inhibitory activity of PARP inhibitor olaparib in several triple-negative breast cancer cells with increased accumulation of DNA DSBs. Our results provide a rationale for the future clinical trials of olaparib combined with SAHA in the treatment of cancers that have an impaired DNA repair ability.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-06.
Abstract P4-16-12: CARE: A pilot study of the effects of short-term fasting on tolerance to (neo) adjuvant chemotherapy in breast cancer patients
Authors: de Groot S , Vreeswijk MPG , Gravesteijn G , Boei JJWA , Jochems A , Houtsma D , Nortier HWR , van der Hoeven KJM , Pijl H , Kroep JR
Published: August 2014

Background:

Preclinical evidence shows that short-term fasting (STS) protects normal cells and makes cancer cells more vulnerable to chemotherapy. This pilot study examines the feasibility and the effects of STS on tolerance to chemotherapy in patients with breast cancer.

Patients and methods:

Eligible patients had histologically confirmed, HER2-negative, early stage breast cancer and adequate bone marrow, liver and renal function. Women receiving (neo) adjuvant TAC courses (docetaxel/adriamycin/cyclophosphamide; day 1, q 3 weeks with G-CSF support at day 2) were randomized to fast 24 hours before and 24 hours after start of chemotherapy or to eat according to the guideline for healthy nutrition. The primary endpoint of the study was to compare neutrophil count after therapy. Secondary endpoints were side effects of chemotherapy, other hematologic counts and chemotherapy-induced DNA damage in leukocytes.

Results:

A total of 13 patients were included of which 7 patients fasted for 48 hours around the chemotherapy infusion (arm A) and 6 patients had a normal diet according to healthy nutrition guidelines (arm B). The median age was 52 years versus 53 years, BMI was 25.5 kg/m2 versus 22.9 kg/m2 and stage III was 43% versus 17% of patients in arms A and B, respectively. Patients were generally motivated to fast and the fasting was well tolerated. Plasma glucose levels were significant lower in fasting patients compared to controls. However, other metabolic parameters showed no significant difference. Fasting did not result in significant differences in neutrophil count or side effects of chemotherapy. Hemoglobin levels and erythrocyte counts after therapy were significantly higher in patients who fasted. Leukocytes of the patients which were isolated at various time points during therapy will soon be analysed for chemotherapy-induced DNA damage and presented at San Antonio.

Conclusion:

This is the first study evaluating the feasibility of 48 hours STS and its impact on side effects of chemotherapy in a homogeneous group of cancer patients. STS was well tolerated and had a beneficial effect on hemoglobin level, but not on experienced side effects. DNA analysis will follow. Larger studies are required to produce more insight into the possible benefits of STS during chemotherapy.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-12.
Abstract P4-19-05: Breast cancer campaign tissue bank human cell culture programme: Generating new biomaterials for breast research
Authors: Gomm JJ , Smith SG , Allen MD , Morgan AS , Haywood LA , Sarper M , Dreger S , Ironside A , Cortes Lopez EJ , Del Rio Hernandez A , Speirs V , Jones LJ
Published: August 2014

Background:

In-vitro model systems provide an important tool in breast cancer research and, over recent years, there have been considerable advances in their construction. However, despite these advances, the majority of models still employ a narrow range of established cell lines, which frequently are not appropriate for the in-vivo cell type they are representing1.

Method:

As part of the Breast Cancer Campaign Tissue Bank, we have developed a cell culture programme which feeds into the main tissue collection and involves the systematic generation of materials from normal, high-risk, cancer-containing and malignant breast tissue in order to make available a breadth of material to the research community.

Following informed patient consent, fresh tissue is retrieved and, using tissue digestion and magnetic bead technology2, purified cell populations are isolated, characterized and frozen down, or further processed for DNA and RNA isolation.

Results:

We have isolated purified populations of luminal epithelial, myoepithelial and fibroblast cells from normal breast, risk-reduction (BRCA1 and BRCA2 mutated) breast and from morphologically normal breast tissue surrounding breast cancer. We have generated matched surround and tumour-associated fibroblasts, as well as matched tumour epithelial and tumour-associated fibroblast cell isolates.

These cells have been incorporated into 2- and 3-D culture models and we have demonstrated successful genetic manipulation, with siRNA gene knockdown and gene over-expression through retroviral transduction. Immortalisation of selected cell populations is currently underway.

Intact organoids and tissue explants have also been generated for in-vitro experimentation. Matched frozen and FFPE samples are available, together with full clinicopathological data. The research community can access these biomaterials via a web-based search portal, with full user support.

Conclusion:

Through this cell culture programme we aim to make available a wider and more appropriate range of materials for breast research, which should allow more clinically relevant model systems to be developed and add value to breast cancer biobanking.

References:

1. Thompson A., et al. Evaluation of the current knowledge limitations in breast cancer research: a gap analysis. Breast Cancer Research 2008; 10: R26.

2. Gomm J.J. et al. Isolation of pure populations of epithelial and myoepithelial cells from the normal human mammary gland using immunomagnetic separation with Dynabeads. Analytical Biochemistry 1995; 226: 91-99.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-19-05.
Abstract P5-01-05: Personalized peptide vaccines in combination with conventional chemo- or endocrine therapy for metastatic breast cancer: A single-arm phase II study
Authors: Toh U , Iwakuma N , Mishima M , Takenaka M , Takahashi R , Koura K , Fujii T , Nakagawa S , Ogo E , Tanaka M , Sasada T , Itoh K , Shirouzu K
Published: August 2014

Background: Immunotherapy using multiple personalized peptide vaccines (PPV) has been reported to be effective for boosting anticancer immunity in patients (pts) with advanced prostate cancer and glioblastoma. The PPVs binding HLA-A2, A24, A3 family and A26 molecules were selected from 31 candidate peptides derived from various tumor associated antigens. Currently, we performed a phase II trial to evaluate the benefit of the PPVs in combination with conventional chemo-or endocrine therapy for pretreated metastatic breast cancer (MBC). Methods: Seventy-one pretreated pts had histologically confirmed measurable MBC and their HLA A molecules matched each of HLA-A2, A24, A26 and A3 family. Pre-vaccination plasma was measured for their IgG levels reactive to each of 31 peptides followed by administration subcutaneously of the four peptides at maximum showing higher levels of IgG in order with schedule of every 1-2 week. The concurrent conventional chemotherapy or endocrine therapy was available for the combination of PPVs. The primary endpoint was anti-tumor immunologic effect and safety, and the secondary endpoints were clinical responses and survival. Levels of IgG reactive to each of the 31 peptides in the pre- and post-treatment plasma at every 6 times of vaccination were measured using LUMNEX system. The CTL responses were simultaneously evaluated using ELISPOT method. Clinical response was evaluated by RECIST criteria. Results: Between May 2009 and December 2012, a total of 71 pts (Luminal-Her2- cases: 39; Her2+cases: 18; Triple negative(TN) cases:14) were enrolled in this study. The median duration of follow-up was 20.7 months. Concurrent chemotherapies (capecitabine, gemcitabine, eribulin, vinorelbine etc.) and endocrine therapies (aromatase inhibitor, toremifen or fulvestrant) were administered to 36 and 24 pts, respectively. Trastuzumab, Laptinib or Bevacizumab was simultaneously used for 13 pts. After 6th vaccination, the IgG reaction and CTL response were identified in 53 of 71 (74.6%) pts and 33 of 63 (52.3%) pts. There were no significant differences between TN cases and the other cases of MBC with regard to immunological humoral and/or cellular responses. No vaccine-related severe adverse events were observed. Clinical response rate of assessable 61 pts was 14.8% (CR 3, PR 6, SD 24, PD 28). The median PFS and OS were 7.6 m and 20.7 m, respectively. In contrast, the PFS and OS of luminal type, Her2 type or TN type were 12, 4.6 or 8.3 m and 24, 15 or 12 m. Conclusions: The phase II PPVs study for MBC demonstrated the promising response and safety, and further studies are essential to identify the clinical benefits of this novel therapy. Clinical trial information: UMIN000001482.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-05.
Abstract P5-01-06: Remodeling the tumor immune microenvironment via intratumoral administration of immunomodulatory agents
Authors: Campbell MJ , Senman B , Esserman LJ
Published: August 2014

Background: Immune dysregulation has been implicated in the poor outcomes associated with high grade and/or triple negative (Tneg) breast cancers. This suggests that a multi-modal approach to stimulating the immune system could be an effective therapeutic strategy for Tneg breast cancer. However, many immunotherapeutic agents demonstrate significant toxicities when administered systemically at high doses. Therefore, we investigated the therapeutic efficacy of local, intratumoral administration of immunomodulators in a mouse model of Tneg breast cancer.

Methods: 4T1, an aggressive, Tneg mammary carcinoma, was implanted orthotopically into syngeneic BALB/c mice. Treatment was initiated when tumors were 0.5-1.0 cm in the largest dimension. We examined various combinations of Toll-like receptor agonists (CpG oligonucleotide [TLR9] and imiquimod [TLR7]) with immunomodulatory antibodies (anti-CD40, anti-CTLA-4, anti-OX40). These agents were administered intratumorally (i.t.), either in PBS or formulated in an in situ depot forming drug delivery system. Growth of the injected tumors was monitored over time. In some experiments, tumors and spleens were harvested 3-10 days post therapy and analyzed for the presence of CD4+ T cells, CD8+ T cells, immunoregulatory T cells (Treg), and myeloid derived suppressor cells (MDSC). To test whether local immunotherapy generates a systemic antitumor immune response, 4T1 tumors were implanted at 2 sites on opposite sides of the mice, with one (left) used as the site of in situ immunomodulation (i.t. injections) and the other (right) observed to assess the systemic antitumor immune response.

Results: Intratumoral administration of various combinations of immunomodulators resulted in complete regression of the injected tumor. These regressions were associated with a reduction in the number of MDSCs in the tumor as well as activation of both CD4+ and CD8+ T cells. Furthermore, local immunotherapy generated a systemic antitumor immune response that inhibited tumors growing at a distant site.

Conclusions: Our findings demonstrate that remodeling the tumor immune microenvironment via intratumoral administration of immunomodulatory agents elicits antitumor immunity that eradicates tumor cells locally as well as systemically at distant sites. The prospect of delivering immunotherapeutics only where it matters most, namely within the tumor microenvironment, is extremely attractive as this would likely be substantially less toxic and more specific than many of the current cancer treatments. Ongoing studies include identifying companion diagnostics that can indicate which patients would benefit most from such an immunotherapeutic approach.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-06.