Canadian Breast Cancer Foundation Research Articles

Omitting Radiotherapy after Breast-Conserving Surgery in Luminal A Breast Cancer

BackgroundAdjuvant radiotherapy is prescribed after breast-conserving surgery to reduce the risk of local recurrence. However, radiotherapy is inconvenient, costly, and associated with both short-term and long-term side effects. Clinicopathologic factors alone are of limited use in the identification of women at low risk for local recurrence in whom radiotherapy can be omitted. Molecularly defined intrinsic subtypes of breast cancer can provide additional prognostic information.MethodsWe performed a prospective cohort study involving women who were at least 55 years of age, had undergone breast-conserving surgery for T1N0 (tumor size <2 cm and node negative), grade 1 or 2, luminal A–subtype breast cancer (defined as estrogen receptor positivity of ≥1%, progesterone receptor positivity of >20%, negative human epidermal growth factor receptor 2, and Ki67 index of ≤13.25%), and had received adjuvant endocrine therapy. Patients who met the clinical eligibility criteria were registered, and Ki67 immunohistochemical analysis was performed centrally. Patients with a Ki67 index of 13.25% or less were enrolled and did not receive radiotherapy. The primary outcome was local recurrence in the ipsilateral breast. In consultation with radiation oncologists and patients with breast cancer, we determined that if the upper boundary of the two-sided 90% confidence interval for the cumulative incidence at 5 years was less than 5%, this would represent an acceptable risk of local recurrence at 5 years.ResultsOf 740 registered patients, 500 eligible patients were enrolled. At 5 years after enrollment, recurrence was reported in 2.3% of the patients (90% confidence interval [CI], 1.3 to 3.8; 95% CI, 1.2 to 4.1), a result that met the prespecified boundary. Breast cancer occurred in the contralateral breast in 1.9% of the patients (90% CI, 1.1 to 3.2), and recurrence of any type was observed in 2.7% (90% CI, 1.6 to 4.1).ConclusionsAmong women who were at least 55 years of age and had T1N0, grade 1 or 2, luminal A breast cancer that were treated with breast-conserving surgery and endocrine therapy alone, the incidence of local recurrence at 5 years was low with the omission of radiotherapy. (Funded by the Canadian Cancer Society and the Canadian Breast Cancer Foundation; LUMINA ClinicalTrials.gov number, NCT01791829.)

87 (PB077) - Optimal dosing of a novel PLK4 inhibitor nested in a phase II study of CFI-400945 in patients with Advanced/Metastatic Breast Cancer (MBC): Canadian Cancer Trials Group (CCTG) IND.237

Background: CFI-400945 is a selective oral inhibitor of Polo-like Kinase 4 (PLK4), a controller of centriole duplication and mitotic progression. CFI-400945 was identified by functional screening of genomically unstable breast cancer (BC). The phase I study escalated doses from 3 to 96 mg/d in a 3 + 3 design; dose-dependent neutropenia (ANC) was noted, and the recommended phase 2 dose (RP2D) declared at 64 mg. IND.237 (NCT01954316) is a phase 2 study in MBC with 3 cohorts, 2 enriched for PTEN status. Enrollment started in 2018 at 64 mg. The initial patients had higher than expected grade 3/4 ANC. Materials and Methods: The primary endpoint of the safety cohort was to confirm the RP2D for CFI-400945. Patients with MBC who met inclusion criteria for CCTG IND.237 were enrolled. A reverse 3 + 3 design was used, and patients enrolled at 64 mg, 48 mg, 40 mg and 32 mg dose levels (DL). In cycle (C)1, patients were treated 7 days on 7 days off for 28 d; for c2 onwards, CFI-400945 was administered continuously. Results: The most common severe adverse event (AE) was dose dependent ANC (table 1). After 80% of the first 5 patients developed grade 3 or 4 ANC, accrual was held while the protocol was amended to add a dose exploration cohort with pharmacokinetics in a reverse dose-finding approach. Based on the frequency of ANC and dose intensity, 32 mg was declared the new RP2D for CFI-400945. There were no non-hematologic severe AEs. PK analysis confirmed similar AUC for the 32 mg DL compared to the PK from the phase I study RP2D.Table 1Frequency of severe neutropenia by DL during C1 and C2 of safety cohort of IND.237DL# PatientsGrade 3 ANC(n(%))Grade 4 ANC(n(%))64 mg51 (20%)3(60%)48 mg65 (50%)2(33%)40 mg82 (25%)2(25%)32 mg60 (0%)0(0%) Open table in a new tab Refinement of the manufacturing process used for IND.237 and subsequent studies incorporated a more stable polymorph of CFI-400945 drug substance had the unexpected property of higher exposure at a given dose and was hypothesized to be due to slightly faster dissolution of the new tablets in the acidic environment of the stomach. Conclusions: The RP2D for CFI-400945 in patients with advanced breast cancer is 32 mg/day. This is 50% lower than the RP2D previously established in the phase I study due to an unexpected increase in exposure resulting from tablets containing a more stable polymorph of CFI-400945 drug substance. Acknowledgements: Sponsored by CCTG. Research supported by a Stand Up to Cancer Canada (SU2C), Canadian Breast Cancer Foundation Breast Cancer Dream Team Research Funding, with supplemental support of the Ontario Institute for Cancer Research through funds provided by the Government of Ontario (Funding Award Number: SU2C-AACR-DT-18-15). Research funding is administered by the American Association for Cancer Research International - Canada, the scientific partner of SU2C Canada. Conflict of interest: Ownership: none Advisory Board: Anthony Lott - Novartis, Eisai, Astra-Zeneca, Ipsen Terry Ng - Novartis, Knight Therapeutics, Boehringer-Ingelheim Arif Awan - AstraZeneca, Eli Lily, Exact Sciences, Exactis, Novartis, Pfizer, Roche Honoraria: Apotex, Roche Rossanna Pezo - Merck, Novartis, Pfizer, Astra Zeneca, Lilly, BMS, Exact Sciences, Myriad Genomics, Mylan Philippe Bedard - Seattle Genetics, Lilly, Amgen, Merck, Gilead Sciences, BMS, Pfizer John Hilton - Bristol-Myers-Squibb (BMS), AstraZeneca (AZ), Pfizer, Novartis, Eli Lilly, Puma, and Genomic Health Jacques Antoun Raphael - Lilly, Merck, AstraZeneca and Novartis. Moira Rushton - Gilead, Pfizer, Viatris David Cescon - Pfizer, AstraZeneca, Novartis, GlaxoSmithKline, Merck, Roche/Genentech, Exact Sciences, Gilead Sciences, Eisai Board of Directors: none Corporate-sponsored Research: Terry Ng - Takeda Oncology Rossanna Pezo - Merck and Novartis Philippe Bedard - Bristol-Myers Squibb, Sanofi, AstraZeneca, Genentech/ Roche, GlaxoSmithKline, Novartis, Nektar, Merck, Seattle Genetics, Immunomedics, Lilly, Amgen, Bicara, Zymeworks David Cescon - Merck, Roche/Genentech, GlaxoSmithKline, Pfizer, Inivata, AstraZeneca, Gilead Sciences Other Substantive Relationships: Terry Ng - Pharma-sponsored education session - ARIAD and Takeda Oncology Arif Awan - Travel: Roche Mark Bray - Full time employee of Treadwell Therapeutics Lesley Seymour & Pierre-Olivier Gaudreau - Funding to CCTG from UHN to partially support trial conduct Moira Rushton - Medical Montior: Pharmamatrix, Edmonton, Alberta David Cescon - Patent (US62/675,228) for methods of treating cancers characterized by a high expression level of spindle and kinetochore associated complex subunit 3 (ska3) gene. note CFI-402257 // CFI-400945 were developed at UHN

DDX41 Is Required to Activate and Modulate cGAS-STING Activation

Upon binding double-stranded (ds)DNA, cGAS is activated and initiates the cGAS-STING-type I interferon pathway; thus, the availability of dsDNA ligand is critical for cGAS activation. DDX41 is a DEAD-box helicase and mutations in DDX41 cause myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, we found that DDX41-knockout (KO) human cells and mice primary cells had reduced type I interferon production after viral infection, which is consistent with previous reports. Unexpectedly, cGAS and STING activation were affected in DDX41 KO cells, suggesting that DDX41 functions upstream of cGAS. The recombinant DDX41 protein exhibited ATP-dependent DNA unwinding activity and ATP-independent strand annealing activity. The MDS/AML-derived mutant R525H had reduced unwinding activities but retained normal strand annealing activity and stimulated greater cGAS dinucleotide synthesis activity than WT-DDX41. Overexpression of R525H in either DDX41-deficient or -proficient cells resulted in higher type I interferon production, indicating that DDX41 inactivates cGAS-STING-type I interferon pathway by unwinding dsDNA to single-stranded (ss)DNA. Our results demonstrate that DDX41 utilizes its unwinding and annealing activities to regulate the homeostasis of dsDNA and ssDNA, which in turn modulates the cGAS-STING pathway; dysregulation of this pathway leads to MDS/AML. Funding Information: This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN 2019-04578) to YZ, the Canada Foundation for Innovation (CFI33364) and Cancer Research Society (CRS-22493) to FJV, the National Institutes of Health (R01-AI-085015) to SRR, and NSERC (RGPIN-2019-05487), the Canadian Breast Cancer Foundation (CBCF-R1636), and the Cancer Research Society (CRS-24139) to YW. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All studies were performed following the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experiments performed with mice in this study were approved by the UIC ACC (protocol no. 15-222). Bone marrow aspirates were collected from 23 patients with AML or MDS who participated in the study according to the protocol approved by the ethics committee on human genome research at Hiroshima University.

Running for the Future: Reproductive Futurity in Canadian Breast Cancer Survivorship Discourse

This article critically examines the proliferation of images of youthful breast-cancer survivors within Canadian Breast Cancer Foundation’s (CBCF) promotional materials, explicating how such images of survivorship are inextricably tied up with the (re)production of gender, sexual, temporal, and citizenship norms. Focusing specifically on the 2013 Run for the Future campaign, I trace how the figure of the prepubescent child and narratives of emerging (hetero)sexuality operate to project a vision of the future, which is marked by the inevitability of both breast cancer and fractured nuclear families. I consider how such imaginations of an unstable future are mobilized to promote participation in the Run for the Cure (the philanthropic event promoted by CBCF’s multimedia campaign). Drawing on insights from queer theory, I highlight how the youthful survivor subject is embedded within a discourse of reproductive futurity in which her nascent citizenship is inextricable from her projected motherhood and heterosexuality. I suggest that the campaign constructs breast cancer as a disease that threatens the integrity and continuance of the heterosexual, nuclear family, and thus constitutes an unavoidable risk that must be addressed by citizens in the name of the future. Ultimately, I argue that participation in the Run for the Cure is styled not as a strategy to protect girls and future women, but rather to safeguard an imagined, desired heterosexual future.

Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis.

BackgroundPreviously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling.MethodsIn vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo.FindingsCACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ).InterpretationCACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling.FundingCanadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research.

Abstract A29: Generation of a novel transgenic mouse model with regulatable β-catenin expression to examine importance of activated β-catenin signaling to long-term maintenance of mammary tumor growth

Abstract Stabilized β-catenin expression is a well described initiator of mammary tumorigenesis in the mouse and elevated nuclear expression has been observed in human triple-negative tumor samples. However, the importance of stabilized β-catenin to continued tumor growth after initiation, and in the context of other driver mutations, has yet to be elucidated. To ascertain the importance of stabilized β-catenin after tumor initiation, we generated a novel transgenic mouse model, utilizing the tet-off system, to control expression of stabilized β-catenin. Expression was tissue restricted through insertion of a stop cassette flanked by loxP sites upstream of stabilized β-catenin and firefly luciferase, which were coexpressed to allow repeated bioluminescent imaging of experimental mice. Insertion of the expression vector was targeted to the Rosa26 locus in R1 embryonic stem cells through homologous recombination. Following aggregation and chimera generation, two founder lines were established. Pups from early litters carrying one allele of regulatable stabilized β-catenin at the Rosa26 locus, but not targeted by Cre, were smaller in size compared with wild-type littermates. They also developed perianal skin lesions around 2-3 weeks of age, with severe necrosis evident by 3-4 weeks of age, requiring the animals to be euthanized. Backcrossing to the desired FVB strain did not alter development of skin lesions, which were observed in offspring from both founder lines. While aberrant gene expression was not observed in these animals, placing breeding females on doxycycline chow through pregnancy, up until pup weaning, eliminated the condition, suggesting a role for the tet machinery in the development of the condition. Maintenance of breeding cages on doxycycline chow allowed for healthy transgenic pups to survive past 3 weeks of age. Initially, six animal cohorts were established, along with control animals, using two different mammary Cre strains, WAP-Cre and MMTV-NLST-Cre, crossed with regulatable, stabilized β-catenin (St-bcatFL) animals, as well as animals expressing mutated p53-R270H. While mammary tumors developed in animals expressing Cre-targeted regulatable St-bcatFL, the penetrance and growth kinetics were lower than observed with other mammary mouse models expressing stabilized β-catenin. As significantly fewer animals developed tumors, we were only able to test the effect of turning off stabilized β-catenin expression following tumor initiation in a small number of animals, with tumor stasis but not regression observed in animals expressing Cre-targeted St-bcatFL, but not in animals expressing both St-bcatFL and p53-R270H. The regulatable St-bcatFL mice were also crossed with animals expressing mutant PIK3CA*E545K and MMTV-NLST-Cre, to examine the importance of stabilized β-catenin to the rapid tumor growth we have observed in mice expressing stabilized β-catenin through deletion of exon 3, and PIK3CA*E545K. While all of the PIK3CA*E545K;Exon3 fl/+; Cre+ animals examined developed multiple mammary tumors by 5 months of age, the PIK3CA*E545K;St-bcatFL;Cre+ mice failed to develop tumors within the same time period. These results indicate that mammary tumor development, including penetrance and growth kinetics, differs significantly between our novel regulatable stabilized β-catenin mouse model and other more aggressive models of stabilized β-catenin. These differences will require alternative methods to determine whether stabilized β-catenin expression is required for the long-term maintenance of tumor growth. Funding for this project was provided by the Terry Fox Foundation, as well as the Canadian Breast Cancer Foundation. Citation Format: Jennifer L. Gorman, Jessica R. Adams, Emma M. Jones, Sean E. Egan, James R. Woodgett. Generation of a novel transgenic mouse model with regulatable β-catenin expression to examine importance of activated β-catenin signaling to long-term maintenance of mammary tumor growth [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A29.

The PAX‐5 transcript is submitted to 3'UTR editing which regulates its oncogenic expression in cancer cells

The Pax‐5 oncogene is a major factor in various cancer lesions such as hematopoietic cancers. Recently, we have also shown that Pax‐5 regulates breast cancer phenotypic transitioning processes (EMT‐MET) leading to the regulation of cancer cell malignancy. To date, the aberrant expression of Pax‐5 in cancer cells is still unknown. Our team has thus turned its attention to the post‐transcriptional regulation of Pax‐5 expression by important regulatory elements such as miRNAs and alternative polyadenylation (APA). Upon our analysis of the Pax‐5 transcript in Pax‐5 bearing cells (breast epithelia and B lymphocytes), we found that not only is the 3'UTR submitted to APA; but also, alternative splicing of the Pax‐5 mRNA 3'UTR. Interestingly, upon rapid amplification of cDNA ends (3'RACE) from polysomal fractions, we found that Pax‐5 3'UTR shortening correlates with increased translation frequency. These observations were also validated using reporter gene assays where various truncated 3'UTRs cloned downstream of a luciferase gene resulted in differentially translational expression. More importantly, we also found that Pax‐5 transcripts characterized with shorter 3'UTRs were also associated with advanced staging of hematopoietic cancer lesions. Our findings identify novel molecular mechanisms, which account for Pax‐5 aberrant expression and function in cancer cells. These findings will further elucidate Pax‐5‐mediated cancer processes and may provide new avenues for therapeutic intervention.Support or Funding InformationThis work was supported by grants from the New Brunswick (NB) Innovation Foundation, the Canadian Breast Cancer Foundation, the Canadian Breast Cancer Society/QEII Foundation, the NB Health Research Foundation and by the Beatrice Hunter Cancer Research InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of Novel Non‐Coding Products of the Pax‐5 Gene and Their Clinical Significance in Lymphoid Cancers

Circular RNAs (circRNAs) are a newly identified class of RNA which are highly expressed and conserved in mammalian cells. With the intrinsic property of being resistant to exonucleolytic activity due to their circular configuration, circRNAs are remarkably stable compared to their linear RNA species counterpart. CircRNAs regulate many biological processes and their aberrant expression is often associated with disease progression. Recently, we have characterized the expression of circRNAs of the potent Pax‐5 oncogene in B cell cancers. In this present study, we set out to identify and establish the expression profiles of Pax‐5 circRNAs in B cell cancers and also, to evaluate the potential of these non‐coding products to act as disease biomarkers. Using RT‐PCR in B cell lines, Pax‐5 circRNAs were amplified with divergent primers. PCR products were then sequenced and aligned for identification and validation. Pax‐5 circRNAs were also evaluated by qPCR from peripheral blood mononuclear cells (PBMCs) from healthy donors and clinical samples. Four circRNA isoforms of the Pax‐5 gene were isolated, sequenced and identified from various B cell models. These latter Pax‐5 circRNA variants consisted of a circular single RNA strand characterized with either exons 2, 3, 4; exons 2, 3, 4, 5; exons 2, 3, 4, 5, 6; or exons 2, 3, 4, 5, 6, 7, 8 of the human Pax‐5 gene. Profiling of Pax‐5 circRNA expression levels in clinical samples reveal an overexpression in chronic lymphocytic leukemia in comparison to other B cell cancer lesions and healthy donors. Altogether, we describe 4 novel gene products (i.e. circRNAs) of the Pax‐5 oncogene in B cell cancers. Preliminary results also support a potential role for Pax‐5 circRNAs as prognostic or diagnostic biomarkers for specific B cell cancers. Further studies are ongoing to validate the correlation between Pax‐5 circRNA levels and disease progression. In addition, the mechanistic elucidation of Pax‐5 circRNAs in cancer processes will also potentially identify new avenues for B cell cancer therapeutic interventions.Support or Funding InformationThis work was supported by grants from the New Brunswick (NB) Innovation Foundation, the Canadian Breast Cancer Foundation, the Canadian Breast Cancer Society/QEII Foundation, the NB Health Research Foundation and by the Beatrice Hunter Cancer Research Institute.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

102P - Functional characterization of mammagliobin-1 isoform in breast cancer

Background: Since 2007, oncologists have been using the GeneSearchTM Breast Lymph Node (BLN) Assay for the detection of metastatic breast cancer. This test detects the presence of Mammaglobin-1 (MGB1) expression in lymph nodes of breast cancer patients. Although MGB1 is overexpressed in 90% of breast cancers, its aberrant expression and function in breast cancer tissue is still misunderstood. We have recently reported the first evidence for MGB1 as potent induction of breast cancer malignancy and disease progression. More precisely, loss of MGB1 expression correlates with a decrease of cell proliferation, spheroid formation, migration, and invasion capacities of breast cancer cells. Concomitantly, we also show that MGB1 expression activates pro-malignant signaling cascades leading to epithelial to mesenchymal transitioning (EMT). More interestingly, we have also discovered new MGB1 gene products that result from transcript editing. The objective of this research is to elucidate the role of MGB1 gene products in breast cancer, we set out to investigate and characterize MGB1 isoforms in breast cancer cell processes. Methods: MGB1 isoforms were identified by 3’and 5’-RACE assays and next generation sequencing using the minion platform. The novel MGB1 gene products were subsequently cloned. Results: In total, we have identified 7 different MGB1 product variants. We strongly believe that each MGB1 product has a specific cellular role, which leads to breast cancer malignancy and disease progression. To perform loss and gain of function experiments, we are genetic engineering breast cancer models that specifically code and express individual MGB1 variants with and without tracking epitope tags. Conclusions: This study will elucidate the molecular and cellular mechanism leading to breast cancer progression. MGB1 variants may provide new avenues for therapeutic or prognostic strategies. Legal entity responsible for the study: N/A Funding: Mitacs Globalink, Baxter and Alma Ricard, Canadian Institute of Health Research, Beatrice Hunter Cancer Research Institute, New Brunswick Health Research Institute, O'Brien Foundation, Canadian Breast Cancer Foundation, Atlantic Canada Opportunities Agency, Université de Moncton Campus Moncton Disclosure: All authors have declared no conflicts of interest.

Prognostic implication of GATA3 gene mutation on survival in invasive ductal carcinoma of the breast.

e13114 Background: GATA3 encodes a transcription factor, which is involved in activation and suppression of genes involved in cell maturation. GATA3 is necessary in the adult mammary gland to maintain the integrity and function of the luminal epithelium. Methods: METABRIC project funded by cancer research UK, the British Columbia cancer foundation and the Canadian breast cancer foundation mapped 173 gene mutations and amplifications in 2,433 primary breast tumors. Retrospective analysis was done for patients with invasive ductal carcinoma of the breast in the age group 30-60; to study the effect of GATA3 mutation on survival. Median survival was obtained from Kaplan-Meier plot, and mortality between groups was compared by Odds ratio (OR). Results: A total of 1500 patients across all age groups had invasive ductal carcinoma. 650 were within the age group 30-60; of which 234 died due to the disease, 360 were alive and 55 died due to other causes. 398 patients (61.2%) tested positive for estrogen receptor (ER) and 521 patients (80.2%) were negative for HER2 (human epidermal growth factor receptor 2). TP53 (50%) and PIK3CA (36%) mutations were more prevalent. GATA3 mutation was found in 79 patients (12.34%); among which, all 79 patients tested positive for ER (100%) and 74 patients (94.9%) negative for HER2. 10 patients (12.7%) died due to the disease, 62 patients (78.5%) were alive and 7 patients (8.9%) died due to other causes. Hence, patients with GATA3 mutations were more likely to survive (OR 4.66; CI 2.33-9.29 p &lt; 0.0001) than patients without the mutation. The median survival for patients with GATA3 mutation (300 months) was also greater than patients without the mutation (219 months). In addition, patients with GATA3 mutation were more likely to be ER positive and HER2 negative. Conclusions: In the mammary gland, GATA3 is required for luminal epithelial cell differentiation. Loss of GATA3 results in de-differentiation to stem cell phenotype. It is found that GATA3 mutation correlates with a better prognosis compared to more common TP53 and PIK3CA gene mutations.

Study of the Mechanisms of Jadomycin Cytotoxicity in Multidrug Resistant Triple Negative Breast Cancer Cells

BackgroundWe previously demonstrated that jadomycins, natural products biosynthesized by the soil bacteria Streptomyces venezuelae, are cytotoxic to drug‐sensitive and multidrug‐resistant breast cancer cells in vitro. Jadomycin cytotoxicity involves the generation of reactive oxygen species (ROS) but the downstream mechanisms contributing to cell death are not completely understood.ObjectiveTo determine if jadomycin anticancer mechanisms involve ROS‐mediated DNA damage and apoptosis in triple negative breast cancer cells in vitro.MethodsMDA‐MB‐231 control (231‐CON) and paclitaxel‐resistant (231‐TXL) breast cancer cells were treated with various concentrations of jadomycin B, F and S or the positive control drug mitoxantrone in the presence and absence of the ROS inducer auranofin or ROS inhibitor N‐acetylcysteine (NAC) for 24 to 36 hours. Western blotting for phosphorylated histone H2AX, a marker of DNA double strand breaks, was used to assess DNA damage. Annexin‐V‐FLUOS and propidium iodide flow cytometric analysis was used to measure apoptosis. The effect of jadomycin treatments on gene expression of topoisomerases IIα and IIβ (Topo IIα and IIβ, respectively) was measured using quantitative PCR. To study the direct inhibition of Topo IIα, Topo IIα decatenation of catenated circular kDNA was measured in the presence and absence of jadomycins.ResultsAll jadomycin treatments and mitoxantrone significantly increased histone H2AX phosphorylation (2.3 – 5.3‐fold) and early apoptosis (1.6 – 3.0‐fold) in 231‐CON and ‐TXL cells versus the vehicle control. NAC and auranofin did not alter jadomycin‐induced DNA damage or early apoptosis. However, auranofin did increase jadomycin‐induced necrosis/late apoptosis (2.7 – 4.5‐fold). Jadomycins caused a significant reduction of Topo IIα and IIβ mRNA expression (4.2 – 8.1‐fold decrease) and directly inhibited Topo IIα in the kDNA decatenation assays.ConclusionsJadomycins induce DNA double strand breaks and apoptosis in drug‐sensitive and multidrug‐resistant MDA‐MB‐231 breast cancer cells. These effects occur independently of ROS generation and may involve gene regulatory and functional inhibition of Topo IIα and IIβ.Support or Funding InformationThe study was funded by the Canadian Breast Cancer Foundation ‐ Atlantic Chapter, the Beatrice Hunter Cancer Research Institute, The Nova Scotia Health Research Foundation and the Dalhousie Pharmacy Endowment. S Hall was a recipient of an Izaak Walton Killam Predoctoral Scholarship. J MacLeod was a recipient of a Beatrice Hunter Cancer Research Institute Cancer Research Training Program Scholarship.

Study of Jadomycin Pharmacokinetics in Balb/C Mice

BackgroundJadomycins are natural product molecules derived from the soil bacteria Streptomyces venezuelae; jadomycins maintain their cytotoxicity in multidrug resistant (MDR) human and mouse breast cancer cell lines that overexpress ATP‐binding cassette (ABC) transporters. While effective in in vitro assays, jadomycins have not been studied in animal models.ObjectiveTo conduct the first in animal pharmacokinetic and safety studies of jadomycins B, S, and F in male and female Balb/C mice.MethodsJadomycins were administered intraperitoneally (IP) to male and female Balb/C mice at a 6mg/kg dose. In one group of mice (n = 5) up to four repeated saphenous vein blood samples (40 mL) were obtained after 15, 30, 60, and 90 minutes with terminal blood collection occurring at 120 minutes in order to characterize absorption and distribution phases. In a second group of mice saphenous vein blood samples were obtained at 2, 4, 6, 8 and 10 hours to characterize terminal elimination. Liver, kidney, lung and brains were collected after 2 hours and then at 8 or 10 hours (males and females respectively) to characterize jadomycin distribution to these tissues. Serum and tissue samples underwent extraction and jadomycin concentrations were then determined by mass spectrometry.ResultsThe concentration versus time profiles of jadomycins B, S and F were similar. They were absorbed rapidly after IP injection with peak concentrations in the 1–10 mM range observed after 15 minutes. This was followed by a rapid distribution phase (t1/2a = 20 – 40 min) and a slower terminal elimination (t1/2b = 1.5 – 2 hours). Jadomycins accumulated in the liver &gt; kidney &gt; lung and were not detectable in the brain. No metabolites were detected and no acute toxicity responses were observed.ConclusionsThese 1st in vivo studies of jadomycins reveal that jadomycins are safe at a 6 mg/kg IP dose and achieve concentrations in mouse serum that are above the IC50s (1–4 μM) for human and mouse breast cancer cells in vitro. Our results indicate that jadomycins are rapidly eliminated as unchanged drug and that two to three times daily dosing will be required for future tumor studies.Support or Funding InformationCancer Research Training Program (CRTP) through Beatrice Hunter Cancer Research Institute, Canadian Breast Cancer Foundation Atlantic (CBCF), Nova Scotia Health Research Foundation (NSHRF), and Dalhousie University

Eligibility Criteria and Genetic Testing Results from a High-Risk Cohort for Hereditary Breast and Ovarian Cancer Syndrome in Southeastern Ontario

Germline mutations in breast and ovarian cancer are rare, with approximately 5% to 10% and 13% being hereditary in origin, respectively. In 2001, the Ontario Ministry of Health and Long Term Care, in an effort to contain costs, defined criteria to determine an individual's eligibility for BRCA genetic screening. We studied a cohort of individuals that have undergone genetic testing at Kingston General Hospital between 2001 and late 2013. We focused on determining whether the 13 risk criteria, defined by an expert working group for the Ontario Ministry of Health and Long Term Care, have performed according to expectations in this cohort. Our findings show that all of the criteria perform well by identifying carriers at the expected 10% rate defined by the guidelines. We demonstrate that loose application of the risk criteria does not further enrich for BRCA variant carriers. Our assessment of the established risk criteria that have been in use in Ontario for more than a decade, provide evidence for their effectiveness, and offer insights into how they may be expanded or improved.

Abstract P6-12-01: Attitudes and practice of breast surgeons towards referring young women with breast cancer (YWBC) for fertility preservation (FP)

Abstract Background: Despite ASCO guidelines (2006, 2013) recommending that young cancer patients be offered referral for FP as soon as possible after diagnosis, the literature consistently shows suboptimal referral rates for YWBC. Surgeons are in a unique position to initiate early FP referral. Surgeon &amp; Patient Oncofertility Knowledge Enhancement (SPOKE) is one of 5 components of the pan-Canadian RUBY research program for YWBC. SPOKE aims to improve breast surgeon FP knowledge and referral rates. The lead surgeon from each of the RUBY sites was previously interviewed about FP. The goal of the current survey was to assess the baseline oncofertility attitudes and practice of the non-lead breast surgeons at those sites. Methods: An online survey taking approximately 10 minutes to complete was developed specifically for this study. In February 2015, an email invitation with a hyperlink to the anonymous survey was sent to all 86 surgeons identified by the 23 lead surgeons. Repeated reminders were sent by the research assistant over 3 months and a final request was sent by the PI. Participants received a $25 gift certificate. Results: A total of 55/86 (64%) surgeons with an average of 15 years' surgical practice completed the survey. 53% were male, 56% were under age 50, and 93% worked at a cancer centre or university-affiliated hospital. Thirty respondents (55%) indicated that more than half of their practice was breast cancer. Twenty (36%) never or rarely initiated a fertility discussion, and 23 (42%) never or rarely discussed FP options with their YWBC. Twenty-two respondents (40%) stated it was the duty of the medical oncology rather than the surgical team to initiate fertility discussions. Only a minority were quite or very familiar with egg freezing (n=10, 19%) and embryo freezing (n=11, 20%), while only 7 (13%) felt comfortable discussing egg or embryo freezing with their patients. Twenty-four (44%) did not know a FP centre in their area to which they could refer. Compared to surgeons who assumed responsibility for fertility discussion, surgeons who did not think FP referral was their clinical responsibility were less familiar with egg freezing (21% vs. 63%, p&amp;lt;.001) and embryo freezing (32% vs. 73%, p&amp;lt;.01), and were less likely to know where to send FP referrals (31% vs. 85%, p&amp;lt;.05). The most common patient factors that surgeons stated would deter them from FP referral were: poor prognosis, need to start chemotherapy urgently, and already having children. A quarter of surgeons said they would be less likely to refer a highly anxious YWBC for FP. Conclusions: Many Canadian breast surgeons are unaware of the importance of early FP referral and nearly half surveyed did not consider FP referral to be their mandate. A majority of these surgeons lack sufficient oncofertility knowledge to feel comfortable mentioning FP options to their patients, and have not created a protocol for FP referral by the surgical team. In the next phases of the SPOKE study, a knowledge translation intervention will be developed and its effectiveness tested. Support: Canadian Breast Cancer Foundation &amp; Canadian Institute of Health Research (OBW139590). Citation Format: Warner E, Yee S, Glass K, Kennedy E, Foong S, Seminsky M. Attitudes and practice of breast surgeons towards referring young women with breast cancer (YWBC) for fertility preservation (FP). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-12-01.

Abstract A09: ACSL4 is a target of the 17beta-estradiol/estrogen-receptor alpha pathway in mammary carcinoma cell lines

Abstract Polyunsaturated fatty acids (PUFA) are important building blocks for cellular membranes and are incorporated into membrane glycerophospholipids to sustain many cellular functions. Newly absorbed PUFA like arachidonic acid (AA) and eicosapentaenoic acid (EPA) are rapidly transformed to acyl-CoAs by members of the acyl-CoA synthetase family (ACSL1, 3, 4, 5 and 6) prior to their incorporation into membrane phospholipids. However, there is little information on the impact of estrogen on PUFA uptake and ACSL expression in estrogen receptor-positive (ER+ve) breast cancer cells. In the current study, the effect of 17 beta-estradiol on PUFA uptake and ACSL expression was measured in (ER+ve) mammary carcinomas cell lines. Stimulation of estrogen-starved ER+ve MCF-7 and T47D mammary carcinomas cells with 17 beta-estradiol resulted in a significant increase in the cellular uptake of exogenous AA and EPA. This was accompanied by a three-fold increase in the expression the ACSL4 isoform measured by western blot in both MCF-7 and T47D cells, but not in immortalized MCF-10A mammary epithelial cells. There was no measured change in the expression of the other ACSL isoforms. ER alpha silencing with siRNA reversed the 17 beta-estradiol-induced ACSL4 expression in both cells lines showing for the first time that hormonal stimulation of ER+ve breast cancer cells is a driver for ACSL4 expression. However, ACSL4 mRNA levels measured by qPCR were unchanged by 17 beta-estradiol treatment, and no change in mRNA stability was measured with 17beta-estradiol treatment as assessed in actinomycin D-treated cells. Conversely, the half-life of ACSL4 protein after treatment with 17 beta-estradiol was extended from 8±0.5 hours in untreated cells to 26±2 hours in 17 beta-estradiol treated cells as assessed following incubation in the absence or presence of cycloheximide. ER alpha silencing with specific siRNA reversed this half-life extension. SHP2 is a tyrosine phosphatase that regulates ACSL4 expression, however the investigation of possible SHP2-mediated changes in ACSL4 in 17 beta-estradiol-treated cells incubated or not with the SHP2 inhibitor NSC87877 showed no difference in ACSL4 expression. Importantly, ACSL4 silencing with siRNAs reversed the 17 beta-estradiol-induced uptake of AA and EPA in both MCF-7 and T47D cells, indicating that ACSL4 was responsible for the enhanced uptake of these PUFA in ER+ve breast cancer cells. Moreover, ACSL4 silencing completely reversed 17 beta-estradiol-induced cell migration as assessed by an in vitro wound healing assay. Overall, these results demonstrate that 17 beta-estradiol enhances the ability of ER+ve mammary carcinoma cells to incorporate PUFA that are important membrane building blocks, via the induction of ACSL4 protein stability. The requirement of enhanced ASCL4 expression for 17 beta-estradiol-induced cell migration suggests that it is a potential therapeutic target in ER+ve mammary carcinoma cells. This work was supported by the Canadian Breast Cancer Foundation, the Canadian Institutes of Health Research and the Canada Research Chairs Program. Citation Format: Anissa Belkaid, Rodney J. Ouellette, Marc E. Surette. ACSL4 is a target of the 17beta-estradiol/estrogen-receptor alpha pathway in mammary carcinoma cell lines. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A09.

Multiple siRNA delivery against cell cycle and anti-apoptosis proteins in breast cancer and normal cells

Event Abstract Back to Event Multiple siRNA delivery against cell cycle and anti-apoptosis proteins in breast cancer and normal cells Manoj Parmar1*, Bárbara E. Arteaga Ballesteros2*, Timothy Fu2*, Remant Bahadur Kc2*, Hamidreza Montazeri Aliabadi3*, Judith C. Hugh4* and Hasan Uludag1, 2, 5* 1 University of Alberta, Faculty of Pharmacy and Pharmaceutical Sciences, Canada 2 University of Alberta, Department of Chemical and Materials Engineering, Canada 3 Chapman University, School of Pharmacy, United States 4 University of Alberta, Department of Laboratory Medicine and Pathology, Canada 5 University of Alberta, Department of Biomedical Engineering, Canada Introduction: Current therapies for breast cancer induce severe side effects that warrant a search for alternative therapies. Short-interfering RNA (siRNA)-based therapeutics directly target and silence specific genes critical for cancer cells, thus holding promise as an effective therapy with less side effects. Given the unregulated cell cycle progression in cancer cells, proteins involved in cell cycle regulation such as spindle assembly checkpoint kinase TTK and cell-division cycle protein 20 (CDC20) are promising targets to stop uncontrolled cell growth. Other targets that might act in conjunction with cell cycle proteins are anti-apoptosis proteins such as survivin [1], which are often up-regulated in transformed cells. We hypothesize that the dual silencing of cell cycle and anti-apoptosis proteins might be exceptionally effective and specific for treatment of breast cancer. Here, we explored the combinational siRNA therapy in breast cancer as well as effects on normal cells in vitro. Methods & Results: Since siRNA is highly labile and anionic, we synthesized a library of cationic and hydrophobic (lipid-modified) polymers from 0.6, 1.2 and 2.0 kDa polyethylenimines (PEI). We screened this library for delivery of CDC20 siRNA in MDA-MB-231 cells, where 1.2 kDa PEI substituted with linoleic acid (PEI-LA) was found to be the most effective [2,][3]. We additionally determined efficacy of 6 siRNAs targeting at different locations of TTK gene; all TTK siRNAs inhibited growth of MDA-MB-231 and MCF7 breast cancer cells (as assessed by MTT Assay), which was further confirmed with the down-regulation of TTK transcripts by qPCR. We then explored the feasibility of combinational siRNA therapy by mixing specific siRNAs with polymers at different weight ratios to form complexes. TTK/CDC20 and CDC20/Survivin siRNA combinations decreased the growth of MDA-MB-231 cells significantly, while only TTK/CDC20 combination inhibited MCF7 cell growth significantly at a relatively low (15 nM each) siRNA concentration (Figure 1). The transcripts of each of these targets were significantly down-regulated in specific siRNA treated cells compared to scrambled siRNA treated cells. The combinational siRNA delivery was also performed in normal breast epithelial MCF10A cells, human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC) to determine the side effects of siRNA treatments using 15 nM each siRNA at different siRNA:polymer ratios. The combinational siRNA therapy at lower ratio (1:2) showed higher side effects of siRNA treatment in these cells compared to higher ratios (1:4, 1:8; Figure 2). Based on relative effect of combinational siRNAs in breast cancer and normal cells, TTK/CDC20 siRNA combination inhibited MDA-MB-231 (33%) and MCF7 (24%) cell growth at 1:4 siRNA:polymer ratio without showing much side effects in MCF10A and HUVEC cells (Figure 2D). However, this combination showed significant side effects in HBMSC cells (33%). Conclusions: This study has indicated improved efficacy of combinational siRNA therapy against cell cycle and anti-apoptosis proteins in breast cancer therapy. The lipid-substituted polymers could serve as a viable platform for delivery of multiple siRNAs against critical targets. However, the siRNA therapy has also showed side effects on non-malignant (normal) cells in vitro and more selective therapies might be needed to target cancer cells solely. Manoj Parmar is a recipient of Women and Children's Health Research Institute (WCHRI) Graduate Studentship Grant and Alberta Innovates: Health Solutions (AIHS) Graduate Studentship.; This study was supported by operating grants from Canadian Breast Cancer Foundation (CBCF) and Natural Sciences and Engineering Council of Canada (NSERC).

White Cashmere Collection 2013

The White Cashmere Collection is Canada’s annual celebration of designing a future without breast cancer. A fund- and awareness-raiser for the Canadian Breast Cancer Foundation (CBCF), the Cashmere Bathroom Tissue Couture collection stars Canada’s leading fashion designers. The first-of-its-kind in the world, and entirely crafted in 100 per cent pure, soft and luxurious sheets of Cashmere Bathroom Tissue, the collection supports the Canadian Breast Cancer Foundation’s vision of a future without breast cancer. More than 125 Canadian designers have participated since 2004. The collection features limited-edition Pink Embossed Cashmere, with twenty-five cents from the sale of every package going directly to CBCF throughout October, Breast Cancer Awareness Month. ©Journal of Professional Communication, all rights reserved.

Abstract P5-07-05: Regulation of system Xc- by signal transducer and activator of transcription 3 and 5 in human breast cancer cells

Abstract In order to survive and proliferate, cancer cells adapt to high levels of oxidative stress by countering the accumulation of reactive oxygen species (ROS) with an increased production of intracellular antioxidant molecules such as glutathione. The cell surface transport system Xc- is a cystine/glutamate antiporter that exports glutamate while importing cystine, thereby mediating levels of cysteine required for glutathione synthesis and the maintenance of cellular redox balance. Transcription factors that regulate key antioxidant defense mechanisms, including system Xc-, may therefore be of therapeutic interest. Recently, signal transducer and activator of transcription (STAT) proteins have emerged as potential targets for the development of novel anti-cancer therapies. In particular, inhibitors of STAT3 and STAT5 may become clinically relevant as anti-cancer agents for breast and brain cancer, as well as leukemia. Interestingly, suppression of STAT3 has been linked with increases in ROS and the induction of apoptosis. Upon activation by phosphorylation, dimerization, and nuclear translocation, STAT proteins transcriptionally regulate diverse target genes by binding to promoter regions containing gamma-activated site (GAS) motifs. The human xCT (SLC7A11) gene encodes the functional subunit of system Xc-. We provide evidence that expression of xCT is regulated by STAT3, and potentially also STAT5, affecting antiporter function in both MCF-7 and MDA-MB-231 human breast cancer cells. Computational analysis of the xCT promoter region revealed the presence of a distal GAS site. Its truncation significantly increased luciferase activity in a reporter assay, with similar increases obtained after treating cells transfected with the full-length xCT promoter construct with various STAT3/5 pharmacologic inhibitors. Knock-down of STAT3 or STAT5A using specific siRNAs produced similar results, suggesting that these STAT proteins act in a transcriptionally repressive manner. We also demonstrated binding of STAT3 and STAT5A to the xCT promoter in MDA-MB-231 cells, which was disrupted by preincubating the cells with specific inhibitors. xCT mRNA and protein levels increased significantly following treatment with STAT3/5 inhibitors. Pharmacologically suppressing STAT3/5 activation also significantly increased glutamate release and total levels of intracellular glutathione. We hypothesize that blocking STAT3/5-mediated signaling induces an adaptive, compensatory mechanism that protects breast cancer cells from ROS by up-regulating Xc- antiporter expression and function. Our findings suggest that targeting system Xc- may synergize with STAT3/5 inhibitors, heightening their therapeutic anti-cancer effects, particularly in conjunction with traditional chemotherapy treatments. This work is supported by the Canadian Breast Cancer Foundation (CBCF). Citation Format: Katja Linher-Melville, Jennifer Fazzari, Patrick Gunning, Gurmit Singh. Regulation of system Xc- by signal transducer and activator of transcription 3 and 5 in human breast cancer cells [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 P5-07-05.

MO‐G‐18C‐07: Improving T2 Determination and Quantification of Lipid Methylene Protons in Proton Magnetic Resonance Spectroscopy at 3 T

Purpose:To improve proton magnetic resonance spectroscopy (MRS) transverse relaxation (T2) determination and quantification of lipid methylene chain (1.3 ppm) protons by rewinding their J‐coupling evolution.Methods:MRS experiments were performed on four lipid phantoms, namely, almond, corn, sunflower and oleic acid, using a 3 T Philips MRI scanner with a transmit/receive birdcage head coil. Two PRESS (Point RESolved Spectroscopy) pulse sequences were used. The first PRESS sequence employed standard bandwidth (BW) (∼550 Hz) RF (radiofrequency) refocussing pulses, while the second used refocussing pulses of narrow BW (∼50 Hz) designed to rewind J‐coupling evolution of the methylene protons in the voxel of interest. Signal was acquired with each sequence from a 5×5×5 mm3 voxel, with a repetition time (TR) of 3000 ms, and with echo times (TE) of 100 to 200 ms in steps of 20 ms. 2048 sample points were measured with a 2000 Hz sampling bandwidth. Additionally, 30 mm outer volume suppression slabs were used to suppress signal outside the voxel of interest. The frequency of the RF pulses was set to that of the methylene resonance. Methylene peak areas were calculated and fitted in MATLAB to a monexponentially decaying function of the form M0exp(‐TE/T2), where M0 is the extrapolated area when TE = 0 ms and yields a measure of concentration.Results:The determined values of M0 and T2 increased for all fatty acids when using the PRESS sequence with narrow BW refocussing pulses. M0 and T2 values increased by an average amount (over all the phantoms) of 31% and 14%, respectively.Conclusion:This investigation has demonstrated that J‐coupling interactions of lipid methylene protons causes non‐negligible signal losses which, if not accounted for, Result in underestimations of their levels and T2 values when performing MRS measurements.Funded by the Natural Sciences and Engineering Research Council of Canada and the Canadian Breast Cancer Foundation – Prairies.NWT

Abstract P4-15-04: Synthesis and characterization of EGFR antibody-mediated tumor targeted “gold nanobombs” for treatment of locally advanced breast cancer

Abstract Purpose: Epidermal growth factor receptor (EGFR) represents an important target for treatment of locally advanced breast cancer (LABC), since about 30% of patients with LABC have triple negative breast tumor, and these tumors are often EGFR-positive. Our aim was to synthesize and characterize a novel form of “gold nanobomb” (GNB) for localized targeted radioimmunotherapy of LABC. GNB uses gold nanoparticles (AuNPs) as a carrier to conjugate both EGFR targeting monoclonal antibody (panitumumab) and the β-particle emitting radionuclide 177Lu. Ultimately, these GNB would be administered locally in LABC for tumor treatment while minimizing normal tissue exposure. Methods: The basic idea for construction of this new-targeted agent is to use different length of poly(ethylene glycol) (PEG) spacers to conjugate antibody and DOTA metal chelators for 177Lu. First, OPSS-PEG-NH2 was used to synthesize OPSS-PEG-DOTA for complexing 177Lu and OPSS-PEG-SVA was used to link antibody. Then these PEG spacers were linked to 30 nm AuNPs to form “GNB”. OPSS-PEG-antibody was analyzed by SDS-PAGE and trinitrobenzenesulfonic acid (TNBSA) assay to determine the degree of amine substitution after PEGylation. A radiometric assay was used to determine the number of PEGylated antibody and DOTA-PEG-OPSS per AuNPs. Specificity of GNB for EGFR and internalization into MBA-MD-468 (1×106 EGFR/cell), MBA-MD-231 (1×105 EGFR/cell) and MCF-7 (1×104 EGFR/cell) was confirmed by confocal and darkfield fluorescence microscopy. Binding assay and cell fractionation study were used to determine the binding affinity to EGFR and the total amount of GNB internalized. Results: Reacting panitumumab with increasing ratios of PEG resulted in an increase in molecular weight from 147 to 250 kDa. This result was in accordance with TNBSA assay where increasing ratio of PEG was associated with increasing lysine substitution (1.4 to 50.9 PEGylated lysine per antibody). PEGylation of 1.4 ± 1.7 lysine per antibody resulted in retention of immunoreactivity and when the immunoconjugates were linked to AuNPs, 8.09 ± 0.38 antibodies were attached to one AuNP. The total number of OPSS-PEG-DOTA per AuNP was estimated to be 812 ± 53. The total size of GNB was 67.3 ± 0.6 nm and there was no particle precipitation or aggregation after incubation in PBS. Competitive binding assay revealed that GNB was targeted strongly to EGFR (Kd antibody: 2.7 ± 0.3 nM; Kd GNB: 5.0 ± 4.9 nM) and darkfield microscopy showed that low PEG to antibody ratio increased the targeting efficiency when compared to high PEG to antibody ratio. Confocal microscopy revealed specific GNB uptake in MDA-MB-468 cells and cell fractionation study demonstrated receptor-mediated internalization of GNB, where the total amount of GNB internalized was about 3.1 and 22.2 fold higher in MDA-MB-468 cells than in MDA-MB-231 or MCF-7 cells. Conclusion: This GNB is very effective in targeting and in internalizing into EGFR, thus the GNB labeled with 177Lu emitting β-particles may be a promising new localized treatment for LABC. We plan to now examine the cytotoxicity of GNB both in vitro and in vivo will be explored for treatment of EGFR positive tumors. Supported by a grant from the Canadian Breast Cancer Foundation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-04.