bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P6-07-03: An exhaustive algorithm for detecting copy number aberrations and large structural variants in whole-genome mate-pair sequencing data
Authors: Asmann Yan W , Wang Chen , Necela Brian M , Chen Xianfeng , Kocher Jean-Pierre A , Maurer Matthew J , Habermann Thomas M , Slager Susan L , Feldman Andrew L , Novak Anne J , Cerhan James R , Perez Edith A , Thompson E  Aubrey
Published: May 2015

Objectives and Rationale: Structural variants (SV) including large copy number aberrations (CNV), translocations, inversions, and large insertions and deletions (INDEL) play a critical role in tumorigenesis and progression. In fact, we now know that tumors can be categorized according to the size of mutations harbored. In several cancers, including ovarian and breast cancer, the large structural mutations, rather than single site mutations, play a dominate role in tumor etiology. Therefore, it's critical to implement reliable algorithm for the detection of structural variants in DNA sequencing data. Mate-pair sequencing is a protocol specifically implemented for detection of the whole-genome level structural variants. It requires less sequencing depth therefor is cost effective, and enables the detection of CNVs, translocations, and inversions simultaneously. However, so far there has been no reliable bioinformatics pipeline for the analyses of the mate-pair sequencing data.

Methods: Our novel algorithm, the SnowShoes-SV, is an exhaustive search algorithm designed specifically for mate-pair DNA sequencing data analyses. It calls the SVs based on disconcordant read pairs. The false SVs are filtered according to the following criteria: (i) the number of the supporting read pairs; (ii) the lack of reads from control data that implicate SV at the same region; (iii) the mappability and uniqueness of the region based on data from the ENCODE project; (iv) consistencies of the mapping orientations of the supporting read pairs; (v) the similar sizes between sequencing library and the two end read clusters.

Results: Using a set of samples previously genotyped by aCGH, the SnowShoes-SV successfully detected all known CNVs and other SVs. It also identified copy number neutral translocations and inversions previously not identified by aCGH. In addition, the algorithm nominated novel SVs which are to be validated by PCR.

Conclusions: SnowShoes-SV is a highly sensitive and specific algorithm for SV detection from the mate-pair DNA sequencing data.

Citation Format: Yan W Asmann, Chen Wang, Brian M Necela, Xianfeng Chen, Jean-Pierre A Kocher, Matthew J Maurer, Thomas M Habermann, Susan L Slager, Andrew L Feldman, Anne J Novak, James R Cerhan, Edith A Perez, E Aubrey Thompson. An exhaustive algorithm for detecting copy number aberrations and large structural variants in whole-genome mate-pair sequencing data [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-03.
Abstract P6-08-31: Linking genotype to clinical outcome in breast cancer by combining NGS and gene chip data
Authors: Gyorffy Balazs , Pongor Lorinc , Kormos Mate
Published: May 2015

Introduction: Next generation sequencing (NGS) provides the possibility to measure mutational status for any part of any gene. However, because of scarce data available to date, linking these mutations to relevant clinical outcome in a large number of patients is not possible.

Aim: Our goal was to combine available genotype data generated by using NGS with gene expression data generated by gene chips to establish a framework to assess the effect of genotype on clinical outcome.

Methods: NGS data generated by the TCGA consortia and publicly available gene chip data obtained from the GEO and EGA repositories were utilized. NGS data was processed using MuTect, SNPeff, GRCh37 and R. RNA-seq data was normalized using DEseq. Gene chip data was MAS5 normalized. Generation of the transcriptomic fingerprint for mutation status was computed by ROC utilizing the RNA-seq data. In the gene chip data, the average expression of significant genes identified was designated as a metagene for the given genotype. Correlation to survival for this metagene was assessed by computing Cox regression and plotting Kaplan-Meier survival plots. Finally, we have set up an online interface to enable running the analysis for any selected gene.

Results: The database contains 332 NGS samples containing mutational status for 22,938 genes and RNA-seq data for 10,987 genes. The gene chip database contains 5,934 patients with 10,987 genes plus detailed clinical characteristics and survival data. We evaluated correlation to outcome for previously identified genes harboring the ten most common somatic mutations in breast cancer. Of these, TP53 (n of mutations out of 332=93, hazard rate=0.51, p<1E-16), AKT1 (n=18, HR=1.6, p=1.6E-15), PIK3CA (n=119, HR=1.5, p=8.5E-12), MAP3K1 (n=20, HR=1.4, p=1.3E-08), CDH1 (n=34, HR=1.3, p=4.4E-07), and RB1 (n=21, HR=1.3, p=7E-06) reached statistical significance while PI3K, PTEN, CDKN1B and GATA3 were not significant or had insufficient number of mutated samples.

Discussion: By connecting genotype to gene expression signature and employing this signature for survival analysis we have set up a pipeline enabling the functional validation of a discovered mutation for any gene in a large breast cancer cohort.

Citation Format: Balazs Gyorffy, Lorinc Pongor, Mate Kormos. Linking genotype to clinical outcome in breast cancer by combining NGS and gene chip data [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-31.
Abstract P6-08-45: Predictive characteristics for extensive nodal involvement in patients with axillary lymph node metastases
Authors: Verheuvel Nicole C , Van den Hoven Ingrid , Ooms Hendrik WA , Tjan-Heijnen Vivianne CG , Roumen Rudi MH , Voogd Adri C
Published: May 2015

Background

Staging of axillary lymph nodes in invasive breast cancer is an important prognostic indicator. Various prediction models have been developed to predict the risk of not having additional axillary metastases in patients with a positive sentinel node, thereby disregarding patients with a positive ultrasound. However, it is important to identify all patients with extensive nodal involvement, defined as 3 or more positive axillary lymph nodes, in whom an axillary lymph node dissection cannot be omitted.

Aim

This study aims to identify factors predicting extensive nodal involvement in the axilla, with the emphasis on the method of axillary staging; ultrasound guided lymph node biopsy versus sentinel node procedure.

Methods

All patients diagnosed with invasive breast cancer in the period between January 2006 and December 2011 at the breast center of the Máxima Medical Center were studied. Univariate and multivariate regression analyses were performed. Variables with a p-value of ≤0.10 in univariate analyses were entered in the multivariate model where a p-value of ≤0.05 was considered statistically significant.

Results

We included 307 cases, representing 306 node positive patients, of whom 178 cases had 1 or 2 positive lymph nodes and 129 cases had 3 or more positive lymph nodes. Multivariate analyses showed that factors as a positive axillary ultrasound (OR=4.513; 95%CI=2.30-8.86), palpability of axillary lymph nodes (OR=2.143; 95%CI=1.04-4.42) and lymphovascular invasion (OR=3.622; 95%CI=1.63-7.81) are significantly associated with extensive nodal involvement in patients with invasive breast cancer.

Conclusion

This study has identified clinically important factors predicting extensive nodal involvement in patients with a positive lymph node biopsy by either a sentinel lymph node procedure or an ultrasound guided lymph node biopsy. Hence, the role of axillary staging by ultrasound should be redefined since it might play an important role in selecting patients with extensive nodal involvement who, in our opinion, may still benefit from axillary treatment.

Citation Format: Nicole C Verheuvel, Ingrid Van den Hoven, Hendrik WA Ooms, Vivianne CG Tjan-Heijnen, Rudi MH Roumen, Adri C Voogd. Predictive characteristics for extensive nodal involvement in patients with axillary lymph node metastases [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-45.
Abstract P6-09-08: Can proton therapy for localized mediastinal Hodgkin's lymphoma reduce second breast cancer incidence?
Authors: Horn Samy R , Pernin Victor , Fournier-Bidoz Nathalie , Kirova Youlia
Published: May 2015

Introduction

Secondary breast cancer (SBC) is a recognized late complication of radiation therapy, after treatment for Hodgkin lymphoma (HL), with reported relative risks as high as 50 with older techniques (Mantle Field). Within the past, it has been estimated that reduction in dose and volume will reduce the incidence of late complications such as secondary neoplasms and cardiovascular disease. We evaluate the impact of mediastinal proton therapy on potential limitation of SBC risk.

Material & methods

For 14 young female patients with early-stage, mediastinal HL, treated with chemotherapy and involved-field raditation therapy (IFRT), we simulated similar treatment plans with conformal radiotherapy (CRT), helical tomotherapy (HT) and proton therapy at the dose of 30 Gy. We report the respective doses to the breasts. Treatment plans were not specifically designed for breast sparing.

Results

Proton therapy significantly lowered the dose to the breasts, with mean doses of 2,76/1,53 Gy to the left and right breast respectively with proton therapy (vs. 4,95/3,88 Gy with HT and vs. 5,56/3,58 Gy with CRT). Proton therapy best limited lower doses (V4Gy and V10Gy) compared to CRT, while HT could limit the higher doses (V20Gy) at the expense of larger volume irradiated at low doses (V4Gy).

Conclusion

Relative reduction in mean doses to the left and right breasts was of 50 and 57% respectively with proton therapy compared to CRT. Relation between radiation dose and SBC seems to be linear, and reduction in SBC should be in such proportion with proton therapy. Proton therapy seems highly interesting for breast sparing after curative treatment for HL, but these results need to be confirmed by individualized risk estimations and prospective trials.

Citation Format: Samy R Horn, Victor Pernin, Nathalie Fournier-Bidoz, Youlia Kirova. Can proton therapy for localized mediastinal Hodgkin's lymphoma reduce second breast cancer incidence? [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-09-08.
Abstract P6-09-09: Body mass index and prognosis after breast cancer diagnosis in Japanese women
Authors: Yamashita Toshinari , Aruga Tomoyuki , Miyamoto Hiromi , Horiguchi Kazumi , Honda Yayoi , Idera Nami , Goto Risa , Kuroi Katsumasa
Published: May 2015

Background: Studies conducted mainly in Western countries have reported a relationship between body mass index (BMI) and prognosis among women with breast cancer. Only a few studies have been conducted in Japan so far because the percentage of high BMI is low. In the present retrospective study, we investigated the associations between BMI and the clinical characteristics and prognosis among breast cancer patients.

Methods: We analyzed 1,744 breast cancer patients who started treatment between 2004 and 2012 at a single hospital in Japan. All patients with ductal carcinoma in situ, male breast cancer as well as metachronous and synchronous bilateral breast cancer were excluded. Median age was 57 years (range 23–91). The number of patients less than 50 years old was 496. World Health Organization BMI classifications were used: Underweight, less than18.5 kg/m2, n=157; Normal, 18.5–24.9 kg/m2, n=1181; Overweight, 25–29.9 kg/m2, n=316; and Obese, more than or equal to 30kg/m2, n=90. The Cox proportional hazards model was used to estimate hazard ratios for recurrence free survival (RFS) in relation to BMI classifications.

Results: Median follow up interval was 4.2 years. During the follow-up period, 126 breast cancer recurrences were observed. BMI classification correlated with clinical tumor size (cT) significantly and BMI classification tended to correlate with lymph node metastases and estrogen receptor (ER) status.

Among patients less than 50 years old, the RFS of those with BMI ≥25.0 kg/m2 was compared to that of patients with BMI <25.0 kg/m2. In multivariate analyses, BMI classification was one of the significant factors (p=0.02) along with lymph node metastases (p=0.0001) and ER status (p=0.04).

However in patients aged 50 years or over BMI category was not a significant factor (p=0.12).

Conclusions: It has been reported that higher BMI is a risk factor for breast cancer recurrence among postmenopausal patients. Our results suggest that higher BMI is also associated with an increased risk of breast cancer recurrence among premenopausal patients. It raises the possibility that maintaining an appropriate body weight improves the prognosis in premenopausal patients after they have been diagnosed.

Citation Format: Toshinari Yamashita, Tomoyuki Aruga, Hiromi Miyamoto, Kazumi Horiguchi, Yayoi Honda, Nami Idera, Risa Goto, Katsumasa Kuroi. Body mass index and prognosis after breast cancer diagnosis in Japanese women [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-09-09.
Abstract P6-12-08: The effects of the BKM120 in combination with herceptin on HER2+ BC cells and BCSCs
Authors: Zhang Jin , Yu Feng , Liu Jingjing , Zhang Xiaobei , Hu Yunhui
Published: May 2015

Introduction: Breast cancer stem cells (BCSCs) are suspected to be responsible for tumour recurrence, metastasis as well as chemo-resistance. Dysregulated PI3K/Akt signaling is implicated in the pathogenesis of a number of breast cancers, including the HER2+ breast cancer. This study evaluated the efficacy of BKM120 monotherapy and BKM120 plus Herceptin in treatment of normal cells and the BCSCs of the HER2+ breast cancer .

Methods:The BCSCs of SK-BR-3 cells were isolated by Serum free cells suspension culture. The impacts of BKM120 monotherapy and BKM120 combined with Herceptin on normal breast cancer cells and BCSCs were assayed by MTT assay, wound-healing assay and plate clone formation assay. The mammosphere-forming efficiency (MFE) of breast cancer cells treated with BKM120 and BKM120 plus Herceptin were also calculated. The nature of the drug interaction of BKM120 and Herceptin was evaluated by using the combination index (CI) according to the method of Chou and Talalay. The impacts of BKM120 monotherapy and BKM120 combined with Herceptin in PI3K/AKT pathway on the normal cancer cells and BCSCs of SK-BR-3 were assayed by Western bloting.

Results: BKM120 showed significant antiproliferative activity in the BCSCs, as well as in normal SK-BR-3 cells. After treated with BKM120, invasion abilities of SK-BR-3 cells was significantly weakened(P<0.01).The colony forming efficiency and the MFE was also decreased compared with the control (P<0.01). Additionally, the pAKT and pS6 expression levels were inhibited. The effects of BKM120 in combination with Herceptin were investigated, which was called Combination Index, showed synergism between the two agents in BCSCs as well as in the normal cells. Furthermore, BKM120 plus Herceptin showed more significantly suppression on following aspects in BCSCs and normal cancer cells : proliferation, invasion ability, colony forming as well as MFE. In addition, the levels of pAKT,pS6 in the BKM120 plus Herceptin group were decreased more obviously than those in control and monotherapy group respectively(p<0.05).

Conclusions:BKM120 can inhibit the growth of normal cancer cells and BCSCs of SK-BR-3, by inhibiting cell proliferation, impairing the invasion abilities and the MFE, and reducing the expression of pAKT(Thr308,S473)and pS6(Thr389).Moreover, these results suggest that the combination of PI3K Inhibitor BKM120 with Herception may provide a novel therapy strategy for HER2+ BC.

Citation Format: Jin Zhang, Feng Yu, Jingjing Liu, Xiaobei Zhang, Yunhui Hu. The effects of the BKM120 in combination with herceptin on HER2+ BC cells and BCSCs [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-12-08.
Abstract P6-16-12: Graded prognostic assessment for triple negative breast cancer brain metastases
Authors: Chi Ming , Alva Venur Vyshak , Abraham Jame , Budd Thomas G , Elson Paul , Ahluwalia Manmeet S
Published: May 2015

Background:

Brain metastases is a serious complication of triple negative breast cancer (TBCBM). We evaluated prognostic factors for overall survival (OS) in contemporary cohort of TBCBM patients treated at a tertiary care institution.

Methods:

With IRB approval, Cleveland Clinic’s database was used to identify BCBM patients treated between 2000 and 2013. OS from the diagnosis of TBCBM was the primary end point. Cox proportional hazards models with stepwise variable selection were used for data analysis.

Results:

One hundred forty three female patients were included for this analysis. Karnofsky performance scale (KPS) was 90-100 in 52 patients (39%), 70-80 in 50 (38%) and <70 in 30 (23%) patients. Sixty eight (49%) patients were treated with whole brain radiation therapy (WBRT), 26 (19%) with stereotactic radiosurgery (SRS), 18 (13%) with WBRT and SRS, 13 (9%) underwent surgery and WBRT, 5 (4%) underwent surgery and SRS. Seventy nine percent (113/143) of patients are reported to have died. Overall survival was 8.3 months (95% C.I. 5.5-10.6). Disease specific graded prognostic assessment (GPA) for breast cancer brain metastases is based on KPS, age at diagnosis, and subtype. By definition triple negative patients fall into the lowest two GPA score groups. GPA was prognostic for survival (p=.05), however separation between groups was variable. Starting de-novo, KPS (but different coding than in the GPA), leptomeningeal disease, and the number of extracranial sites of disease at the time of diagnosis of TBCBM were found to be independently prognostic for survival (proportional hazards model, stratified by when brain metastases were diagnosed, and a stepwise selection algorithm). A new GPA was formulated by assigning "points" (weights) to the levels within each of these three factors that are proportional to the regression coefficient estimates in the final model and then adding the total number of points present.

Prognostic Factors Impacting Survival of TBCBMFactorNumber of pointsHazard RatiopKPS0.31 (0.21-0.48)<.000180-1003  </= 700  No. Extra-Cranial Mets0.42 (0.28-0.64)<.00010 or 12  >/= 20  Leptomeningeal Disease0.43 (0.26-0.71)0.001No2  Yes0  

The new GPA consists of three groups: unfavorable, intermediate and favorable with OS of 2.7, 9.1 and 13.6 months respectively.

New GPAGPANumber of pointsNumberMedian OS ( months)PFavorable736 (27%)13.6 Intermediate4-559 (45%)9.1 Unfavorable</= 336 (27%)2.7     <0.0001

Conclusions:

A novel GPA for TBCBM is proposed.

Citation Format: Ming Chi, Vyshak Alva Venur, Jame Abraham, Thomas G Budd, Paul Elson, Manmeet S Ahluwalia. Graded prognostic assessment for triple negative breast cancer brain metastases [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-16-12.
Abstract S3-07: 16 year long-term follow-up of the IBIS-I breast cancer prevention trial
Authors: Cuzick Jack , Sestak Ivana , Cawthorn Simon , Hamed Hisham , Holli Kaija , Howell Anthony , Forbes John F
Published: May 2015

Background: Several randomised clinical trials have shown the benefit of tamoxifen in healthy women to reduce their risk of breast cancer. Here, we report the blinded median 16 year follow-up of the IBIS-I trial to update the long-term prevention of breast cancer with tamoxifen treatment.

Methods: 7154 pre- and postmenopausal women were randomised to receive daily 20mg tamoxifen (N=3579) or matching placebo (N=3575) for 5 years. The primary endpoint of this analysis was the occurrence of breast cancer (invasive and ductal carcinoma in situ (DCIS)). Secondary endpoints included overall mortality, other cancers, and breast cancer specific mortality. Cox proportional hazard models were used to assess occurrence of breast cancer and survival. All statistical tests were two-sided.

Results: After a median of 16.2 years (IQR 14.4 to 17.7) of follow-up, a total of 589 breast cancers have been reported (tamoxifen: 246 (6.9%) vs. placebo: 343 (9.6%)). Tamoxifen reduced the incidence of all breast cancer overall by 29% (HR=0.71 (0.60-0.83), P<0.0001) (Figure 1). Invasive ER-positive (ER+) breast cancers were reduced by 35% (HR=0.65 (0.53-0.80), P<0.0001) (Figure 1), but no effect was seen for invasive ER-negative (ER-) breast cancers (HR=1.06 (0.71-1.58), P=0.8). A non-significant 30% reduction in DCIS was seen with tamoxifen (36 vs. 51, HR=0.70 (0.46-1.07); P=0.1). The overall risk reduction was similar in years 0-10 (HR=0.71) and years 10-20 (HR=0.70). Similar effects were seen in pre- and postmenopausal women (HR 0.71 vs. 0.71). All-cause mortality was non-significantly increased in women randomised to tamoxifen (173 vs. 158, OR=1.10 (0.88-1.38), P=0.4). The excess in deaths with tamoxifen is smaller than in the 96 month update. No differences in breast cancer mortality was seen (24 tamoxifen vs. 27 placebo; OR=0.89 (0.49-1.60), P=0.7). A non-significant increase in other cancers than breast were reported by women on tamoxifen (350 vs. 315, OR=1.12 (0.95-1.32); P=0.2). Specifically more endometrial cancers (28 vs. 17), non-melanoma skin cancers (108 vs. 85), and lung cancer (32 vs. 20) were found in those randomised to tamoxifen.

Conclusion: This updated analysis of the IBIS-I trial confirms the significant reduction in breast cancer occurrence with tamoxifen in the post-treatment follow-up period. These results indicate tamoxifen has a long-term preventive effect on invasive ER+ breast cancer in both pre- and postmenopausal women.

Citation Format: Jack Cuzick, Ivana Sestak, Simon Cawthorn, Hisham Hamed, Kaija Holli, Anthony Howell, John F Forbes. 16 year long-term follow-up of the IBIS-I breast cancer prevention trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S3-07.
Abstract SF1-2: Improving access to innovative anticancer medicines: a clinicians viewpoint
Authors: Schellens Jan H. M.
Published: May 2015

For most forms of cancer there is high unmet need. This is especially the case for advanced cancer. Improved understanding of mechanisms of unresponsiveness to currently available treatments and identification of novel drugable targets have boosted anticancer drug development, often leading to so-called breakthrough medicines, i.e. drugs that can dramatically improve cure rate and survival. The monoclonal antibodies (mAb) trastuzumab and rituximab and the tyrosine kinase inhibitor (TKI) imatinib, developed in the recent past, deserved this qualification. These drugs have not only changed the standard of practice, they also served as template for new drug development leading to EMA & FDA approval of >30 novel mAbs and TKIs for cancer indications since the introduction of rituximab in 1998. However, this does not guarantee swift patient access to new medicines nor to equal access for all indicated patients. Also, some drugs with high anticancer potential in well defined cancer populations simply seem to be stuck in development. One of the examples of missed opportunities is the group of the PARP inhibitors (PARPi): first patient included in phase I was in the year 2005 and now almost a decade later there is still no availability worldwide for well characterized populations with BRCAm, including breast and ovarian cancer, of PARPi treatment. Clearly, there is room for improvement, for which there is a range of good opportunities: shorten time to registration, shorten time to reimbursement, improve patient selection, limit to well defined subsets and expand later, setup global biomarker networks, setup global well controlled access programs and lastly take more risk. These opportunities will be discussed and examples will be given.

Citation Format: Jan H. M. Schellens. Improving access to innovative anticancer medicines: a clinicians viewpoint [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr SF1-2.
Abstract A021: Loss-of-function screen for breast tumor initiating cells reveals PRC1 dependence
Authors: Chung Chi-Yeh , Gaspar-Maia Alexandre , Mullokandov Gavriel , Bosch-Gutierrez Almudena , Farias Eduardo , Brown Brian , Bernstein Emily
Published: May 2015

Breast tumor-initiating cells (BTICs) are a subset of cells within breast tumors thought to be more tumorigenic than non-BTICs and possess both self-renewal and differentiation potential. Gene expression and functional studies have demonstrated a correlation between BTICs and high-grade tumors, poor patient prognosis, and therapy resistance; however, the mechanisms underlying BTIC regulation remains poorly understood. Because epigenetic alterations play a key role in breast cancer progression and in stem cell regulation, we hypothesized that epigenetic factors mediate BTIC function. Using a mouse MMTV-Myc mammosphere (MS) culture system, which we showed to be enriched in BTICs by in vivo tumorigenicity assays and gene expression studies, we performed a pooled shRNA library screen coupled to next generation sequencing (NGS). Using a library of ~450 shRNAs targeting 60 epigenetic regulators, our screen identified multiple members of the Polycomb Repressive Complex 1 (PRC1) as top candidates, with shRNAs targeting PRC1 members significantly depleted in MS cells, but not in control bulk cells. This strongly suggests that PRC1 is required for MS/BTIC function. We have validated that knockdown of PRC1 members reduces MS formation and BTIC ‘markers’ in MMTV-Myc cells and human MDAMB-231Luc cells in vitro, and are currently performing in vivo tumorigenesis assays. Further, RNA-Seq and ChIP-Seq is underway to decipher the mechanisms by which PRC1 mediates BTIC regulation. In accordance with our screen, The Cancer Genome Atlas (TCGA) reports several members of PRC1 to be overexpressed in breast tumors, and we are further exploring the clinical relevance of PRC1 members in breast cancer patient samples.

Citation Format: Chi-Yeh Chung, Alexandre Gaspar-Maia, Gavriel Mullokandov, Almudena Bosch-Gutierrez, Eduardo Farias, Brian Brown, Emily Bernstein. Loss-of-function screen for breast tumor initiating cells reveals PRC1 dependence. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A021.
Abstract A027: Endothelial-like properties of triple-negative claudin-low breast cancer cells promote tumor-vascular interactions and tumor permeability
Authors: Harrell J. Chuck , Pfefferle Adam , Zalles Nicole , Prat Aleix , Fan Cheng , Khramtsov Andrey , Olopade Olufunmilayo , Troester Melissa , Dudley Andrew , Perou Charles
Published: May 2015

The different intrinsic subtypes of human breast tumors vary in rate and location of metastatic dissemination. In this study, we aimed to determine if tumor-associated vascular properties could help to explain the differences observed in metastagenicity. We used endothelial and breast cancer cell line models to develop gene expression signatures that measured vascular quantity and vascular proliferation. We tested these signatures, amongst other published endothelial signatures, and found that the genomic programs that measured vascular quantity, vascular proliferation, and a VEGF/Hypoxia-signature were the most highly expressed in claudin-low and basal-like tumors. We then tested the vascular signatures on five human breast cancer datasets (n>3,000) and identified two vascular signatures that added prognostic information to intrinsic subtype classification. Next, microvessel density (MVD) scores were obtained from a set of 70 tumors which were also subjected to gene expression profiling. Interestingly, several of the vascular signatures added metastasis-predicting information in addition to MVD (p<0.05). Similar to the tumor data, claudin-low cell lines also expressed high levels of vascular gene signatures as compared to luminal cell lines. Interestingly, pure claudin-low cell lines, and subsets of claudin-low-like cells within established basal-like cell cancer cell lines, exhibited endothelial-like morphology when culture on Matrigel. In vivo xenografts of claudin-low tumors, but not luminal tumors, extensively perfused injected contrast agent through paracellular spaces and non-vascular tumor-lined channels. Taken together, the endothelial-like characteristics of the cancer cells, combined with both the amount and the physiologic state of the vasculature, contribute to breast cancer metastatic progression. We hypothesize that the genetic signatures we have identified highlight patients that should respond most favorably to anti-vascular agents.

Citation Format: J. Chuck Harrell, Adam Pfefferle, Nicole Zalles, Aleix Prat, Cheng Fan, Andrey Khramtsov, Olufunmilayo Olopade, Melissa Troester, Andrew Dudley, Charles Perou. Endothelial-like properties of triple-negative claudin-low breast cancer cells promote tumor-vascular interactions and tumor permeability. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A027.
Abstract A040: Id-1 increases mammary basal stem cell activity and tumorigenesis by activating the Wnt/c-Myc pathway
Authors: Shin Dong-Hui , Lee Jeong-Yeon , Jang Ki-Seok , Min Kyueng-Whan , Jang Si-Hyong , Oh Seung Hyun , Kong Gu
Published: May 2015

Inhibitor of differentiation/DNA binding (Id)-1 is a crucial regulator of mammary development and breast cancer progression. Here, we demonstrate that Id-1 induces mammary tumorigenesis by increasing normal and malignant mammary stem cell (MaSC) activities. MaSC expansion and increased self-renewal and in vivo regenerative capacity of MaSCs were observed in the mammary glands of MMTV-Id-1 transgenic mice. Id-1 maintains the MaSC-enriched basal cells, but not the luminal cell lineage, in the mammary glands. Furthermore, Id-1 induces ductal hyperplasia and mammary tumors with high expression of basal markers in MMTV-Id-1 transgenic mice. It also increases breast cancer stem cell populations and activity in human breast cancer lines. In experiments with mouse primary mammary epithelial cells and human cancer cells, the effects of Id-1 on normal and cancer stem cell activity were shown to be mediated by the Wnt/c-Myc pathway. Collectively, these findings indicate that Id-1 regulates mammary basal stem cells by activating the Wnt/c-Myc pathway, thereby contributing to basal marker-positive breast tumor development.

Citation Format: Dong-Hui Shin, Jeong-Yeon Lee, Ki-Seok Jang, Kyueng-Whan Min, Si-Hyong Jang, Seung Hyun Oh, Gu Kong. Id-1 increases mammary basal stem cell activity and tumorigenesis by activating the Wnt/c-Myc pathway. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A040.
Abstract A076: Deficiency of Fes tyrosine kinase expression correlates with delayed mammary tumor onset in a HER2/Neu overexpressing transgenic mouse model
Authors: Zhang Connie Shengnan , Greer Peter A.
Published: May 2015

Fes (a.k.a. Fps) is a cytoplasmic protein tyrosine kinase that is highly expressed in breast epithelial cells during lactation, as well as in vascular endothelial and myeloid cells. Retrovirally encoded oncogenic alleles of Fes are associated with tumors in chickens and cats and cause tumors in transgenic mice; however, a role for Fes in human cancer has not been established.

Using an orthotopic mouse mammary engraftment model, we previously found that Fes deficiency correlated with reduction in engrafted tumor growth rates, lung metastasis, and circulating tumor cells. Reduced vascularity and fewer infiltrating macrophages suggested the tumor microenvironment of Fes-deficient mice was less supportive of tumor growth and metastasis. In co-culture with tumor cells, Fes-deficiency in macrophages correlated with a reduced ability to promote tumor cell invasive behavior. These results suggested a tumor promoting role of Fes kinase in breast cancer through roles in cells of the tumor microenvironment.

In a transgenic mouse model of breast cancer driven by an activated HER2/Neu allele expressed in the mammary epithelium, we observed delayed tumor onset in Fes-deficient mice compared to wild-type mice. However, there was no difference in tumor growth rates.

Taken together, these observations argue that Fes inhibition might provide therapeutic benefits in breast cancer, by attenuating tumor-associated angiogenesis and the metastasis-promoting functions of tumor-associated macrophages, or by delaying breast tumor onset in women with HER2 overexpression.

Note:This abstract was not presented at the conference.

Citation Format: Connie Shengnan Zhang, Peter A. Greer. Deficiency of Fes tyrosine kinase expression correlates with delayed mammary tumor onset in a HER2/Neu overexpressing transgenic mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A076.
Abstract A113: Chronic stress promotes lymph node metastasis through beta-adrenergic regulation of lymphangiogenesis
Authors: Le Caroline P. , Pimentel Matthew A. , Kim Corina , Nowell Cameron J. , Karnezis Tara , Chai Ming G. , Herald Marco , Stacker Steven A. , Sloan Erica K.
Published: May 2015

Lymphangiogenesis drives breast cancer metastasis to lymph nodes. However, the physiological pathways that regulate lymphangiogenesis and lymph node metastasis are yet to be defined. We have previously shown that chronic stress increased metastasis of primary tumour cells to lymph nodes via beta-adrenergic receptor signaling. Consistent with a critical role for beta-adrenergic signaling in cancer progression, recent clinical studies found that blockade of beta-adrenergic activation protected women with triple negative breast cancer from distant metastasis.

To investigate if the beta-adrenergic pathway promotes metastasis by impacting lymphangiogenesis, we explored the effect of restraint stress or pharmacological activation of beta-adrenergic pathways in an orthotopic xenograft model of triple negative breast cancer. To physiologically activate beta-adrenergic signaling, mice were subjected to 2 hours of daily restraint for 7 days prior to tumor cell inoculation and 14 days thereafter. Serial bioluminescence imaging revealed chronic stress reduced time to lymph node metastasis and increased tumor cell load in draining lymph nodes. Stress increased the expression of lymphangiogenic factor VEGF-C and its receptor Vefgr-3 and increased lymphangiogenesis within primary tumors. Pharmacological activation of beta-adrenergic signaling with isoproterenol similarly increased lymphangiogenesis and metastasis.

Prostaglandin-mediated dilation of lymphatic collector vessels was recently linked to increased breast cancer metastasis. We found stress increased expression of COX-2, the rate limiting enzyme in prostaglandin production, and increased dilation of lymphatic vessels that link primary tumors to draining lymph nodes. Treatment with COX-2 inhibitor celecoxib reversed stress-induced lymph vessel dilation and similarly blocked stress-enhanced lymph node metastasis and tumor lymphangiogenesis.

Together, these data show that beta-adrenergic receptor signalling may be an important effector pathway of stress-enhanced lymphangiogenesis and LN metastasis and that beta-blockade of lymphangiogenesis may be an important mechanism in protecting women with breast cancer from the adverse effects of stress biology.

Citation Format: Caroline P. Le, Matthew A. Pimentel, Corina Kim, Cameron J. Nowell, Tara Karnezis, Ming G. Chai, Marco Herald, Steven A. Stacker, Erica K. Sloan. Chronic stress promotes lymph node metastasis through beta-adrenergic regulation of lymphangiogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A113.
Abstract A118: The mTORC2 component Rictor directs branching morphogenesis and survival in the mammary epithelium
Authors: Morrison Meghan M. , Hicks Donna , Cook Rebecca S. , Brantley-Sieders Dana M.
Published: May 2015

The intracellular serine/threonine kinase mTOR functions in two distinct complexes that integrate many cellular cues to regulate diverse processes in development and disease. Raptor-dependent mTOR Complex-1 (mTORC1) mediates cell growth and metabolism, whereas the less well-studied Rictor-dependent mTOR Complex-2 (mTORC2) promotes cell survival and cytoskeletal organization. Previous studies link mTORC1 to malignant transformation of mammary epithelial cells (MECs). The role of mTOR and the relative contributions of mTORC1 versus mTORC2 in postnatal mammary morphogenesis, however, remain unclear. We report that mTORC2 ablation via Rictor loss impaired branching morphogenesis, MEC survival, MEC migration, and mTORC2-mediated signaling through Akt and Rac1-GTPase in vivo and ex vivo. Constitutively active Rac1 (CA-Rac1) rescued branching, invasion, and survival of mTORC2-impaired MECs, but CA-Akt did not. These effects were recapitulated by rapamycin treatment. Surprisingly, genetic mTORC1 disruption via Raptor gene targeting did not affect ascinar mammary morphogenesis in organoids or in vivo, but did cause decreased signaling through the mTORC1 effector ribosomal protein S6 and transiently slowed cellular proliferation. These data demonstrate the distinct, non-overlapping properties of mTORC1 and mTORC2 during post-natal mammary development, roles which may be preserved in breast cancers.

Citation Format: Meghan M. Morrison, Donna Hicks, Rebecca S. Cook, Dana M. Brantley-Sieders. The mTORC2 component Rictor directs branching morphogenesis and survival in the mammary epithelium. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A118.
Abstract A130: Progesterone expands a hormone-dependent progenitor population in luminal breast cancers
Authors: Fan Huaping , Horwitz Kathryn B.
Published: May 2015

More women die of Luminal, hormone-dependent, breast cancers than any other types. These tumors are estrogen (ER) and progesterone (PR) receptor-positive. Luminal cancers can recur many years after diagnosis, and when they do, they are just as deadly as rapidly-recurrent Basal cancers. Cytokeratin 5 (CK5) is a marker of triple negative Basal breast cancers. We have models in which Luminal breast cancer cells grown as xenografts in ovx'd mice or as 3D colonies in hormone-free or estrogen (E)-containing media are ER+PR+CK5–. However, if E is supplemented with progesterone (P), an ER–PR–CK5+ Basal-like subpopulation expands; we term these cells “Luminobasal”. Detailed IHC shows heterogeneity within the E+P-induced Luminobasal population, with rare cells that are ER+PR+CK5+; hence “Double Positive”. Similar “Double Positive” cells can be detected in clinical Luminal tumor samples stained for PR and CK5. Luminal cells in E+P colonies were FACS-sorted into CK5+ and CK5– subpopulations. RNA was extracted and subjected to gene expression profiling. The gene list was analyzed for cell surface markers unique to the CK5+ cells that might be useful for live-cell FACS sorting. Several were tested, and one marker is of considerable interest. FACS analysis shows that these Double Positive cells are rare (0.12%) in E, but increase to 6.63% under E+P. The cells are confirmed PR+CK5+ by IHC. Importantly, these PR+CK5+ cells form mammospheres indicating their self-renewal capacity. In the absence of hormones, the cells within the mammospheres differentiate into the heterogenous subpopulations that characterize Luminal disease, including PR+CK5–, PR–CK5+ and PR+CK5+ cells. Studies of hormone dependence and tumor-forming potential are ongoing. We speculate that Double Positive cells are progenitors of Luminal tumor-cell heterogeneity.

Citation Format: Huaping Fan, Kathryn B. Horwitz. Progesterone expands a hormone-dependent progenitor population in luminal breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A130.
Abstract B022: cDNA microarray analysis of genes associated with reduced annexin A6 expression in invasive breast cancer cells
Authors: Sakwe Amos M.
Published: May 2015

Recent studies have shown that the calcium-dependent membrane binding protein, annexin A6 (AnxA6) is involved in cell proliferation, membrane dynamics, calcium fluxes across the plasma membranes and cell-cell and cell extracellular matrix interactions and cell motility. Together with our recent observation that AnxA6 is down regulated in breast cancer, this suggests that AnxA6 may influence the expression of a very diverse set of genes. We therefore, investigated changes in gene expression profiles associated with AnxA6 depletion in invasive breast cancer cells. To do this we generated AnxA6 depleted BT-549 cells with varying extents of AnxA6 down regulation. Two of the cell lines designated BT-A6sh2 and BT-A6sh5 as well as pGIPZ transfected control cells were used in the cDNA microarray analysis. Analysis of the microarrays revealed that a total of 558 genes were significantly down-regulated of which 38 were down regulated by at least 2.0-fold and p < 0.05. Meanwhile, 625 genes were significantly up-regulated and 33 were up regulated by at least 2.0-fold and p < 0.05. Interestingly, the greater the extent of AnxA6 depletion the greater the number of genes that were up or down regulated. Among the up regulated genes were RASGRF2, CDH2, JAG1, PCDH10 and PCDH7. Among the down regulated genes were FNDC1, CD24 SLC4A4, CALB2, CSPG4 and ST8SIA6. Interestingly, LGALS3 and CDH3 were among several other genes that were up regulated while EGFR, a predictive marker of basal-like breast cancer, was among the genes that were down regulated following >80% depletion of AnxA6. Validation of some of these genes by quantitative RT-PCR suggests that AnxA6 influences Ras signaling, cadherin-mediated cell-cell interactions, solute transport, cell-extracellular matrix interactions, calcium signaling and more interestingly, cancer stem cell phenotypes. Down-regulation of CD24 and up-regulation of N- and P-cadherin suggest that reduced AnxA6 expression as previously shown in breast cancer tissues also facilitates the transformation of tumor cells into CD24-low mesenchymal stem cell-like phenotypes. These data suggest that reduced AnxA6 expression in invasive breast cancer is accompanied by modulation of a wide range of genes that influence several aspects of breast cancer progression, some of which are potentially useful targets for prognosis and therapeutic intervention.

Citation Format: Amos M. Sakwe. cDNA microarray analysis of genes associated with reduced annexin A6 expression in invasive breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B022.
Abstract B047: Cyclin-dependent kinase 2 regulates androgen receptor activity in estrogen receptor negative breast cancer
Authors: Proctor Luke A. , Hanson Adrienne R. , Butler Miriam S. , Tilley Wayne D. , Moore Nicole L.
Published: May 2015

There are limited treatment options for estrogen receptor (ER)-negative breast cancer, a disease subtype that is highly aggressive and associated with poor clinical outcome. The androgen receptor (AR) is expressed in approximately 30% of ER-negative breast cancers and is emerging as a viable therapeutic target. AR activity is influenced by numerous intracellular signalling pathways which post-translationally modify the AR protein to finely regulate its stability and transcriptional output. This study is investigating the functional interaction between cyclin dependent kinases (CDKs) and AR in breast cancer. We hypothesize that CDK2 is required for optimal AR activity and androgen-regulated cell growth in ER-negative breast cancer cells and that inhibition of CDK2 may be an effective approach to reduce ER-negative breast cancer cell growth. AR-positive, ER-negative MDA-MB-453 breast cancer cells were treated with the androgen 5α-dihydrotestosterone (DHT) in the presence or absence of the CDK2 inhibitors roscovitine, NU6102 or CVT-313. The effects on AR target genes as well as total and phosphorylated AR were assessed by quantitative RT-PCR and Western blotting while MTT assay, trypan blue dye exclusion and flow cytometry were used to examine cell proliferation and viability. DHT induced expression of the AR target genes FKBP5, c1ORF116, SEC14L2 and RANBP3L however each of the CDK2 inhibitors completely blocked the stimulatory effect of DHT on these genes. The CDK2 inhibitors also reduced the ability of DHT to stabilize AR protein and induce Ser81-AR phosphorylation. Basal and DHT-induced cell proliferation was blocked by the CDK2 inhibitors, and this was associated with cell cycle arrest and cell death. The results suggest that AR requires CDK2 for optimal transcriptional activity and proliferative effects in breast cancer cells. This may have important therapeutic implications for ER-negative, AR-positive breast cancers.

Citation Format: Luke A. Proctor, Adrienne R. Hanson, Miriam S. Butler, Wayne D. Tilley, Nicole L. Moore. Cyclin-dependent kinase 2 regulates androgen receptor activity in estrogen receptor negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B047.