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Background: The PI3K pathway is dysregulated in the majority of triple-negative breast cancer (TNBCs). Contrary to the theory of oncogene addiction, single agent inhibition of the PI3K pathway in TNBC has had only modest activity. Our group has demonstrated preclinically that when PI3K is inhibited, an immediate compensatory up-regulation of the Wnt pathway occurs. The Wnt pathway is known known for its role in cancer metastases and can confer resistance to initial PI3K inhibition. Simultaneous dual targeting of both pathways is highly synergistic against TNBC models in vitro and in vivo. We have initiated a Phase I clinical trial using Gedatolisib (PI3K/mTOR inhibitor) and PTK7-ADC (Wnt pathway) for patients with metastatic TNBC (NCT03243331). Gedatolisib is a pan-class I isoform PI3K/mTOR inhibitor, and PTK7-ADC is an antibody-drug conjugate against the cell-surface PTK7 protein (Wnt pathway co-receptor) with an Auristatin payload. PTK7 is an attractive second target due to its up-regulation after PI3K inhibition and its known overexpression in TNBC. Further data has shown that the PTK7-payload, Auristatin, is in itself synergistic with Gedatolisib. The combination of using both of these drugs suggests a unique concept of “double synergy”. Where Gedatolisib increases the expression of the target of PTK7-ADC leading to one mechanism of synergy, and the Auristatin payload on PTK7-ADC is synergistic with Gedatolisib providing a second mechanism. Study Design: This is an open-label, Phase I, dose-escalation study with a 3 + 3 cohort design. The trial will enroll 12-18 patients. 3 cohorts of at least 3 patients will receive Gedatolisib (weekly) & PTK-ADC (q3w) at 110mg+1.4mg/kg, 180mg+1.4mg/kg, and 180mg+2.8mg/kg dose levels. Eligibility Criteria: This trial enrolls patients with metastatic triple negative (ER-, PgR-, HER2-) or low estrogen expressing (ER and PgR Objectives: The primary objective is to evaluate the safety of Gedatolisib plus PTK7-ADC. The secondary objective is to evaluate efficacy as determined by objective response rate, clinical benefit at 18 weeks, and progression free survival (PFS). Exploratory objectives will evaluate efficacy in patients with genomic aberrations in the PI3K pathway; and association of tumor DNA, RNA, plasma and circulating tumor cell sequencing with clinical efficacy to identify putative biomarkers. Correlative Sciences: We are collecting matched pre-/post-treatment tumor biopsies and serial blood samples to determine biomarkers of clinical response to inform subsequent trials. We plan to evaluate: 1) PI3K activity; 2) genomic aberrations in the PI3K pathway; 3) baseline PTK7 expression; 4) PTK7 upregulation after Gedatolisib treatment; and 5) mutations in plasma circulating tumor DNA. Supported by the BCRF, 100 Voices of Hope, Catherine Peachey Foundation, and Pfizer. Citation Format: Radovich M, Solzak JP, Hancock BA, Storniolo AMV, Schneider BP, Miller KD. An initial safety study of gedatolisib plus PTK7-ADC for metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-06-02.

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of the estrogen and progesterone receptors and lack of HER2 over-expression. ENMD-2076 is an orally bioavailable small molecule inhibitor of Aurora and angiogenic kinases with pro-apoptotic and antiproliferative activity in preclinical models of TNBC. Methods: This two institution, single-arm, two-stage, phase II clinical trial enrolled patients with locally advanced or metastatic TNBC refractory to 1-3 prior lines of chemotherapy in the advanced setting. Patients had ECOG PS ≤ 1, measureable disease by RECIST 1.1 and no evidence of brain metastasis. Patients were treated with ENMD-2076 250 mg PO daily with continuous dosing in 4-week cycles until disease progression or unacceptable toxicity occurred. The primary end point was 6-month clinical benefit rate (6-CBR) and secondary endpoints included time to progression (TTP), PK profile, safety and biologic correlatives in archival and fresh serial tumor biopsies in a subset of patients. Results: Between July 2012 and October 2016, 41 patients were enrolled (median age 54; range 30-73; female 40; male 1). Patients received a mean 1.7 prior lines of chemotherapy for locally advanced unresectable or metastatic disease and 80.5% received prior neoadjuvant or adjuvant chemotherapy (N=33). Thirty-six patients were evaluable per protocol for the primary efficacy analysis. Five patients (12.2%) were not included in the efficacy analysis due to: adverse events (AE) leading to discontinuation prior to objective efficacy assessment (N=3), not meeting eligibility criteria on day 1 (N=1) and withdraw of consent in cycle 1 (N=1). The study proceeded to the second stage of enrollment based on observing three 6-CBR events in Stage 1 (N=18 patients). The 6-CBR in the overall trial was 16.7% (95% exact CI: 6%-32.8%; 2 patients with PR and 4 patients with SD > 6 mos). The median duration of response or clinical benefit in these patients was 32 weeks (8 cycles). 4-CBR was 27.8% (95% exact CI: 14%-45.2%). Dose reduction occurred in 8 patients (20%) for fatigue, hypertension and proteinuria. The most common grade 3 treatment-related adverse events were hypertension (37.5%) and fatigue (10%). One patient experienced grade 4 hypertension. Analysis of serial tumor biopsies prior to and following 2 weeks of ENMD-2076 (N=8 patients), demonstrated a treatment-induced decrease in cellular proliferation (Ki-67) and microvessel density (CD34) as assessed by IHC. Immunofluorescence performed on a subset of samples demonstrated an increase in p53-family member expression following treatment, consistent with changes observed in preclinical TNBC patient-derived tumor xenograft models. Conclusions: ENMD-2076 has durable clinical activity in a subset of patients with pretreated, advanced or metastatic triple-negative breast cancer. Predictive biomarker development using archival and fresh tumor tissue is underway. Exploration of lower doses of ENMD-2076 in future clinical trials may improve tolerability. Citation Format: Diamond JR, Eckhardt SG, Pitts TM, van Bokhoven A, Aisner D, Gustafson DL, Capasso A, Elias AD, Storniolo AM, Schneider BP, Gao D, Tentler JJ, Borges VF, Miller KD. Clinical safety and efficacy of the aurora and angiogenic kinase inhibitor ENMD-2076 in previously treated, locally advanced or metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-16.

Recent data demonstrating a correlation between lymph node positivity at the time of detection, and the probability of disease recurrence even decades post detection only solidifies the principle that the detection of breast cancer prior to lymph node metastasis can appreciably better clinical outcomes. Although radiologic methods have greatly improved early detection and remain the mainstay for detection, molecular assays to complement existing strategies will reduce number of false positives as well as enhance detection in cases that preclude conclusive diagnosis with radiologic techniques. Normal breast biology is routinely studied using tissues from reduction mammoplasty or normal tissues adjacent to tumor (NATs). However, studies have shown histologic abnormalities in reduction mammoplasty samples and DNA methylation and gene expression abnormalities in NATs due to “field” effects of the tumor. To interrogate the differences between normal breast and NATs as potential early detection markers, we created a tissue microarray (TMA) comprising breast tissues of 100 age-matched healthy women from the Komen Tissue Bank (KTB) and tumor-NAT pairs from 100 women (a total of 300 samples). Approximately 50% of women in each set were of African American (AA) ancestry and the remaining was of European decent. The TMAs was curated as such, because of our recent findings on ethnicity-dependent differences in breast stem-luminal progenitor-mature cell hierarchy. TMA was analyzed for ZEB1, an oncogenic transcription factor that is central to cell fate and stemness, and estrogen receptor alpha (ERα) and FOXA1, which are expressed predominantly in hormone-responsive mature luminal cells. ZEB1 expressing cells were localized to surrounding ductal structures of the normal breast, whereas ERα+ and FOXA1+ cells were located within the ductal compartment. KTB-normal of AA women contained significantly higher levels of ZEB1+ cells compared to KTB-normal of Caucasian women (CA). We observed only marginal increases in ZEB1+ cells in NATs or tumors of AA women. By contrast, in CA women, both NATs and tumors compared to KTB-normal contained higher levels of ZEB1+ cells. The unique localization pattern external to the ductal structures, as well as intrinsically higher expression in AA women suggest that ZEB1+ cells serve not only as stem cells from which cancers may originate but could also contribute to the microenvironment conducive for ductal tumor progression leading to aggressive and early onset of breast cancer as observed in AA women. Conversely, KTB-normal of AA showed modestly higher FOXA1 expression compared to CA women, and further, FOXA1 levels were declined in NATs of AA but not CA women. ERα levels did not change in any of our analyses, pointing to the specificity of ethnicity-dependent changes in this TMA. We also noted ethnicity-dependent variations in the levels of CD8+ T cells, PD-1+ immune cells and PD-L1+ cells but not CD68+ macrophages in NATs, suggesting distinctive immune environment in NATs. This comprehensive approach will not only serve as a platform to develop tumor-adjacent “normal” tissues as molecular markers for early detection but also provides a molecular basis for aggressive breast tumor in AA women. Citation Format: Nakshatri H, Kumar B, Burney H, Cox ML, Jacobson M, Sandusky G, D'Souza-Schorey C, Storniolo AM. Harnessing the distinctive properties of tumor-adjacent tissues to develop ethnicity-dependent biomarkers of breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-03-11.

Functional modeling of breast epithelial hierarchy and stromal-epithelial cell interactions has been difficult due to inability to obtain sufficient stem-progenitor-mature epithelial cells and stromal cells. The recently developed epithelial reprogramming assay has partially overcome this limitation, allowing propagation of epithelial cells with stem, luminal progenitor and mature cell features. However, characterizing stromal cells using this assay is difficult because irradiated fibroblasts which can be difficult to distinguish from stromal cells are needed as feeder layer. A recent study demonstrated expansion of airway basal stem cells without a feeder layer through pharmacologic inhibition of TGFβ/BMP/SMAD signaling. We sought to develop this method for culture and expansion of cells from normal and cancerous breast samples. With appropriate modifications to growth media, we were able to obtain normal and stromal cells from breast biopsies of healthy women. The expanded cell population included CD10+/EpCAM- basal/myoepithelial cells, CD49f+/EpCAM+ luminal progenitor cells, CD49f-/EpCAM+ mature luminal cells, CD73+/EpCAM+/CD90- rare endogenous pluripotent somatic stem cells, CD73+/CD90+/EpCAM- mesenchymal stem cells, ALCAM (CD166)+/EpCAM+ cells, CD44+/CD24- cells, CD44+/CD24+ cells and ALDFLUOR+ stem/luminal progenitor cells. Epithelial cells were KRT14+, KRT19+ or both further documenting heterogeneity within epithelial cell population. We have extended this technique to grow breast epithelial cells from high-risk patients including BRCA1 mutant-carriers, tumor-adjacent normal and tumor cells from the same patient, pleural effusions and liver metastasis from breast cancer patients. Phenotypic characterization showed differences in the differentiation state of adjacent-normal and tumor cells. Tumor cells from pleural effusions showed remarkable phenotypic heterogeneity with a fraction of these cells expressing estrogen receptor. The assay described here, therefore, is versatile and provides resources to model epithelial-stromal interactions under normal and cancerous conditions as well as for genomics and screening of drugs to target metastasis on an individual level. Susan G. Komen for the Cure and Department of Defense supported this work. Citation Format: Nakshatri H, Ananappa M, Prasad MS, Kumar B, Liu Y, Storniolo AM, Miller KD, Bhat-Nakshatri P. Dual TGFβ/BMP inhibition allows in vitro expansion of multiple cell types from normal and cancerous breast [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-08.

Background Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. This ignorance has been the consequence of the lack of access to richly annotated, high quality normal breast specimens. The Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB) was established expressly to remedy this deficiency. The KTB is a repository of specimens from volunteer donors with no clinical evidence of breast malignancy. The Bank's mission is to make available specimens that will enable an understanding of the developmental biology of the normal breast, to provide insight into breast oncogenesis, and to provide a normal control for breast cancer research. The purpose of this presentation is to increase the awareness of this unique and rich research resource and to actively solicit the use of its specimens. Methods: The KTB has been prospectively banking fresh frozen breast tissue since mid-2006. Coincident with the tissue donation two tubes of blood are obtained, which are processed for lymphocyte DNA, serum and plasma. These specimens are richly annotated with detailed information regarding the donors’ reproductive history, medical history, family history, and medications. Standard Operating Procedures have been constructed so as to control, limit and identify potential sources of bias. All of this information is recorded in an Oracle-based, searchable database. Results: As of June 2011, the KTB and its predecessor bank, Mary Ellen's Bank, have available fresh frozen breast tissue (10 gauge cores) from 1469 donors; formalin-fixed, paraffin-embedded tissue from 1055; DNA from 7507; serum from 2382; and plasma from 3771 donors. The KTB has also established 28 epithelial and 33 stromal cell lines from the cores; 4 of the epithelial cell lines have been immortalized using hTERT. Donors range in age from 18–86 years of age. 9% of donors to the KTB describe themselves as Hispanic/Latino. 5.2% of donors are Black or African-American. Using the Gail Risk Model, there is a bimodal distribution of life-time breast cancer risk among the donors: the largest peak is at 10% and a smaller one at 18%. Conclusions: The KTB is a unique and invaluable research resource which is now open for business and accessible to researchers across the globe. We encourage researchers to avail themselves of this unique tissue resource and to also acquaint themselves with other sources of healthy breast tissue, i.e., the Love/Avon Army of Women [http://www.armyofwomen.org/]. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-21-02.

Background:The BRCA1 and the BRCA2 proteins play a role in DNA repair and confer genomic stability to the cell. Identifying BRCA mutation carriers has become an important tool for prevention as well as guiding therapy in cancer patients. We proposed to test the hypothesis that gene expression analysis of peripheral whole blood can reliably detect these mutations. Materials and methods: Following IRB approval, 10cc of blood was collected from 36 women (BRCA1 (n=8), BRCA2 (n=9), Hereditary breast cancer without BRCA (FAM) (n=7), sporadic breast cancer (SPO) (n=11)). 3 of BRCA1 and 5 of BRCA2 samples were from women without cancer. Following RNA extraction (using the method described by Beekman et al) and quality assessment, Illumina® Whole-Genome DASL™ microarray (Human Ref-8 BeadChips) analysis was performed. The raw data was normalized and analyzed using Partek® Genomic Suite. Differentially expressed genes were identified using ANOVA analysis. Geneset specific supervised analysis was performed to visualize the inherent similarities and differences in the gene expression amongst different groups for 1) DNA repair and 2) Immune-system-related genes. Ingenuity Pathway Analysis (IPA) was performed to interpret the data in the context of biological processes, pathways and networks. Results: Twenty-nine of the 87 immune-related genes were up-regulated in BRCA1 and BRCA2 groups compared to SPO or FAM groups; these included IL7R, CD53, CD2, CD48 and HLA-DRA. Twenty-five of the 79 DNA repair genes were up-regulated in BRCA1 and BRCA2; these included FANCC, RAD51L3, MSH2, MSH6 and PCNA. In IPA analysis, the comparison of BRCA1 vs. REST (BRCA2 + FAM + SPO) showed a strong immunologic signal, with the top altered biological processes including “Immunologic disease”, “Infection mechanism”, “Immune cell trafficking” and “cell-mediated immune response “. The top 5 canonical pathways also reflected a similar pattern and included “iCOS-iCOSL Signaling in T Helper Cells”, “OX40 Signaling Pathway”, “Calcium-induced T Lymphocyte”, “Apoptosis Regulation of IL-2 Expression in Activated and Anergic T Lymphocytes” and “Protein Ubiquitination Pathway”. When BRCA2 was compared with the REST (BRCA1 + FAM + SPO), a much weaker signal was noted with none of the canonical pathways being significantly altered. PAM analysis showed that a set of 16 genes could differentiate the BRCA patients from the rest with an error rate of 5%. Further validation of this geneset is being performed. Conclusion: Gene profiling in whole blood may offer an easy, reliable and inexpensive way to identify patients with BRCA mutation. Further studies are currently underway to validate our results in a larger patient population. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-13-01.

Purpose: Lymphedema is a significant long-term complication of primary therapy for breast cancer. VEGF has a major impact on vascular permeability and lymphangiogenesis. In a prior case control study, we found a significant increase in serum VEGF-C (median 6895 vs. 5349 pg/mL, p=0.0016) in women with lymphedema compared to women without lymphedema suggesting inhibition of VEGF-C as a potential therapeutic strategy. Methods: Pts with significant unilateral lymphedema were treated with pazopanib 800 mg/day continuously. Baseline assessments include arm volume, interstitial fluid pressure (IFP), extracellular fluid volume by lymphometer (ECF), quality of life (QOL) and plasma VEGF-C,-D, and R3. IFP was measured serially for 24 hours after the first treatment; arm volume, ECF, QOL, and plasma VEGF-C/D/R3 are assessed every 4 weeks. Results: Ten pts were enrolled. Median duration of lymphedema was 3.5 years (0.7-8.2) Median time since surgery was 5.1 years (1.0-14.2); median time since radiation (n=9) was 4.7 years (0.4-8.8). Complete IFP data are available in 7 pts. Baseline IFP was significantly higher in the affected compared to unaffected arm (mean 5.73 vs. -1.78 mmHg; p=0.0005). Mean IFP in the affected arm decreased an average of 76% 24 hours after initiation of pazopanib (5.73 vs. 1.56 mmHg; p=0.0061). Toxicity was greater than anticipated with grade 3 fatigue (n=3), arthralgia (n=2), and anorexia (n=1); three pts discontinued therapy prior to the first post-treatment assessment. Five pts met the definition of response with a ≥25% reduction in excess arm volume four weeks after initiating treatment. ECF significantly (p=0.014) decreased as well though the mean difference in excess arm volume did not significantly change (p=0.06). Conclusions: Preliminary data supports the hypothesis that VEGF plays a role in the development and/or persistence of lymphedema after local therapy for breast cancer. Pazopanib acutely decreases IFP, leading to a decrease in ECF and modest improvement in arm volume but results in significant toxicity at the dose used on this study. Subsequent studies will explore a lower dose of pazopanib in this population. This study was supported by the Breast Cancer Research Foundation with pazopanib supplied by GSK. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-14-02.

Background: Triple-negative breast cancer (TNBC) has been plagued by the absence of targeted therapies. Discovery of therapeutic targets in TNBC has in part, been hampered by an inadequate understanding of the transcriptional biology of the normal breast as an optimal comparator. Using next-generation sequencing, we embarked on a study to compare the transcriptomes of TNBC and normal breast to comprehensively identify novel targets by analyzing all full length transcripts expressed in these tissues. Methods: Normal breast tissues from healthy pre-menopausal volunteers with no history of disease were procured from the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. To eliminate bias from stromal tissue, normal tissues were laser capture microdissected for ductal epithelium. cDNA libraries from 10 TNBC tumors and 10 normal breast tissues were sequenced on an Applied Biosystems (AB) SOLiD3 sequencer using 50bp fragment runs. For gene expression, mapping of reads to the genome was performed using the AB BioScope 1.2 Pipeline and outputs imported into Partek Genomics Suite for analysis. In Partek, mapped reads were cross-referenced against known genes from the UCSC database followed by statistical comparison of RPKM values for each gene between TNBC and normal. Dimensionality reduction analyses (PCA & Hierarchical clustering) and identification of Novel Transcribed Regions were also performed in Partek, whereas construction of gene networks was performed using Ingenuity Pathway Analysis. To identify gene fusions, partially mapped reads were interrogated utilizing a novel algorithm that searched for reads spanning exons from two different genes. Fusions that were supported by at least 3 reads (of which 2 had to be unique) were considered candidates and were subsequently validated. Results/Discussion: Sequencing produced 1.1 billion reads equaling 57.3GB of data of which 36.0GB (63%) mapped to the human genome. In comparing RPKM values between TNBC and Normal, we report 7140 RefSeq Genes, 22 pre-miRNAs, 109 lincRNA exons, and 15 ultraconserved regions that were differentially expressed between these tissues (FDR Conclusion: We report an extensive comparison of the transcriptomes of TNBC and normal ductal epithelium. We identified numerous genes previously unknown to be dysregulated in TNBC that can be utilized for therapeutic discovery. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD01-08.

Background: Gene expression data from pancreatic cancer, histologically normal tissue adjacent to the cancer and normal pancreas reveals that adjacent normal has already acquired a number of transcriptional alterations and is not, therefore, an appropriate baseline for comparison with cancers. (Gadaleta et al., 2011) The purpose of this study was to determine if this is also the case for breast cancer and, if so, to identify the differences in gene expression between adjacent normal and normal breast. Methods: RNA-Seq data from breast cancer and adjacent normal was downloaded from the TCGA (The Cancer Genome Atlas) data portal. The epithelia from 20 frozen tissue cores from healthy premenopausal donors to the Susan G. Komen for the the Cure® Tissue Bank at the IU Simon Cancer Center were microdissected and the RNA isolated. RNA-seqeuncing was carried out using the Life Technologies SOLiD Platform. RPKM gene expression values from TCGA and sequencing of the Komen normal tissues were merged, quantile normalized, and batch effect corrected. Normalization and differential gene expression was performed using Partek Genomics Suite. Results: Principal component analysis (PCA) reveals complete separation between adjacent normal and healthy normal breast tissue. Setting a maximum FDR (false discover rate) of 5%, 2239 genes are differentially expressed between adjacent normal and healthy normal. Ingenuity pathway analysis reveals that the Fos, Jun and TGFbeta pathways are active in the adjacent normal. Conclusions: Tissue adjacent to a primary breast cancer is not normal when using healthy breast tissue as a comparator. As RNA-Seq data is digital, it is possible to quantify the changes in gene expression starting from healthy normal to normal adjacent to tumor to tumor. Increasing and decreasing gene expression values provide clues to the fundamental molecular changes occurring in histologically normal appearing adjacent tissue. The differences in gene expression we have identified are some of the earliest changes in breast carcinogenesis and provide insight into the etiology of this disease and, potentially, its prevention. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-03-02.

Background: The cell of origin of metaplastic carcinoma of the breast (MCB) is an enigma. MCB comprises less than 4% of all breast cancers, and is a member of the subtype of breast cancer referred to as Triple Negative Breast Cancer (TNBC). It is aggressive with a prognosis worse than that that for invasive ductal carcinoma NOS (not otherwise specified), as well as other TNBCs. Identification of the histogenesis of MCB is likely to provide clues to its oncogenesis. Differentiation along non-epithelial lineages is also observed in benign lesions of the breast. Adenomyoepitheliomas, thought to arise from myoepithelial cells, have been observed to contain areas of cartilaginous, sebaceous, squamous, and osseous differentiation. Methods: Healthy woman volunteers from central Indiana were invited to donate breast tissue to the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. Starting from the 10 gauge tissue cores, 28 normal mammary epithelial (HME) and 33 normal stromal (HMS) cell lines were established using an organoid isolation method after digestion with enzymes for 24 hours. The HME cell lines were characterized by immunohistochemistry (IHC). Ploidy was determined by karyotype and Interphase FISH mapping with centromere probes for Chromosomes X and 17. Cellular morphology was observed both on two-dimensional and in three-dimensional culture systems. The HME cells were subjected to FACS analysis using multiple antibodies including CD24, CD44, Muc1, CD49f, and EpCAM. Results: 96.9% of early passage cells are diploid. The HME cells express vimentin, CK 5/6, p63, CD 10, CK 18, and HER-1 when grown on two dimensional plastic surfaces. Cells placed in the center of a sandwich of Matrigel® uniformly form spheres 37mm-325mm in diameter. Hematoxylin and eosin staining of the formalin-fixed and paraffin-embedded sections of these spheres reveal keratinized squamous differentiation. When the cells are grown on Laminin, Collagen IV, or Fibronectin surfaces multiple cell types are observed including osteoclasts, distinguished by the presence of Tartrate Resistant Acid Phospatase; and chondrocytes, confirmed by staining with Alcian Blue. Other cells with a spindle-shape and cytoplasmic vacuoles turn a dark reddish-brown color when stained with Oil Red O, characteristics of adipocytes. In other areas of the culture, the cells form a syncitium and they express the protein MyoD, a marker of immature muscle. Finally, there are numerous cells with long, dendritic processes. These cells express Nestin, a marker of neural stem cells; glial fibrillary acidic protein (GFAP), expressed by mature astrocytes; and beta-III tubulin produced by differentiated neurons. Using FACS, the HME cells were found to be CD49f positive and EpCAM negative. Multiple nucleoli were confirmed using anti-Nucleostemin IHC. Conclusions: Phenotypic plasticity is common to all the HME cell lines characterized to date. Differentiation into cells of mesodermal and ectodermal origin, CD49+/EpCAM- by FACS, and the presence of multiple nucleoli suggest that the isolated cells are a multipotent/stem cell residing in the normal adult breast. These cells, through a series of yet to be elucidated events, may be the cells of origin of MCB. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-02.

Background: Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established expressly to address and remedy this deficiency. The KTB acquires and banks normal breast tissue, that is, breast tissue from volunteer donors with no clinical evidence of breast malignancy. This tissue is NOT from reduction mammoplasties or from histologically normal tissue adjacent to a malignancy. The breast is one of the most complex genetic organs within the body. This is because the expression of its genes is under the control and influence of the hormonal milieu present in the circulating plasma, which changes as a function of age; for premenopausal women as a function of the menstrual cycle; and as a consequence of pregnancy. Therefore, there is unlikely to be a singular “normal” breast. We propose to produce a molecular encyclopedia of the normal breast which covers the entire spectrum of normal: puberty to menopause, low risk to high risk, nulliparous and parous. Materials and Methods: The epithelial compartment of fresh frozen tissue from 10 premenopausal donors to the KTB, 5 women who were in the follicular phase of the menstrual cycle and 5 who were in the luteal, was isolated using laser capture microdissection. Total RNA extracted from the cells was subsequently depleted for ribosomal RNA. RNA was sequenced on an Applied Biosystems SOLiD3 sequencer using 50bp runs. Reads were mapped to the human genome. Whole blood was collected at the time of tissue donation and uniformly processed into serum. Results: RNA sequencing of the 10 samples produced 596 million reads of which 386 million (62%) mapped to the human genome. Setting the p-value at Proliferating Cell Nuclear Antigen (PCNA), nucleosome assembly genes and genes involved with mitosis have greater expression during the luteal phase of the menstrual cycle. Genes associated with development, e.g., NOTCH2, PAX3, DKK3 and TWIST1, are more abundantly expressed during the follicular phase. Many of the differentially expressed genes have been implicated in breast oncogenesis. Conclusions: The Komen Tissue Bank has completed the first ever next-generation transcriptome sequencing of epithelial compartment of ten normal human breast specimens. This work has produced the most comprehensive catalog to date of the differences in the expression of protein encoding genes, pre-miRNAs, lincRNA exons, UCRs and novel transcribed regions as a function of the phase of the menstrual cycle. Additionally, this effort has identified a relatively significant number of genes whose expression is very likely under the control of estrogen. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-01.

Background: The neoadjuvant setting provides an ideal opportunity to explore the impact of sunitinib alone and in combination with paclitaxel on the functional status of tumor vasculature as indicated by changes in tumor interstitial pressure (IFP) and circulating progenitor cell (CPC) subsets.Methods: Patients(pts.) with newly diagnosed stage Ic-IIIc breast cancer were treated with sunitinib monotherapy (100 mg Day 1; 37.5 mg D2-14) prior to the initiation of paclitaxel (80 mg/M2 D1,8,15 every 28 days x 4 cycles) with sunitinib (25 mg/d). IFP and CPC subsets were assessed at baseline and after sunitinib monotherapy. IFP was measured in three separate areas of the tumor using a micropressure transducer catheter; mean and highest IFP recorded were analyzed. Improvements in poly-chromatic flow cytometry allowed distinction of pro-angiogenic (pCPC = Live/AC133+CD34bright/CD31+CD45dim\), from non-angiogenic (nCPC = Live/AC133-CD34bright/CD31+/CD45dim\), CPCs that cannot be quantified separately using previously reported protocols1. Baseline CPC subsets were also compared to age and gender matched healthy controls.Results: From July 2008 to May 2009, 14 pts were enrolled; baseline and post-sunitinib IFP and CPC data are available for 12 and 11 pts respectively. The median age was 48 (range 32-69). Mean pretreatment tumor diameter was 3.2 cm. Sunitinib monotherapy was generally well tolerated; two patients developed grade III toxicities including hypertension (n=1) and hand-foot syndrome (n=1). Sunitinib significantly decreased mean IFP (18.87 mmHg vs. 6.38 mmHg; p=0.002); similar results were obtained when only the maximum IFP was considered (22.32 mmHg vs. 8.36 mmHg; p=0.003). pCPC frequency was not different between pts and healthy controls (0.099% vs. 0.076%, p=0.24) but the p:nCPC ratio was significantly higher in pts compared to healthy controls (3.26 vs. 1.46; p=0.001) suggesting a shift toward vascular damage and/or active angiogenesis. Both pCPC frequency (0.099 vs. 0.022; p=0.001) and p:nCPC ratio (3.26 vs. 0.78; p=0.001) significantly decreased after sunitinib monotherapy. Analysis of paclitaxel + sunitinib is too early (n=4).Conclusion: Sunitinib, through its effects on VEGF-mediated vascular permeability, markedly reduces tumor IFP. In addition sunitinib profoundly reduces pCPCs that are thought to be important for angiogenesis in human cancers. Accrual is ongoing (planned N=40). Analysis of IFP and CPC subsets after paclitaxel + sunitinib as well as additional correlates will be presented.1 Duda et al. (2007). Nat. Protocol 2, 805-810. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 202.

9523 Background: Lymphedema is a significant long-term complication of primary therapy for breast cancer. Anecdotally, several patients (pts) with metastatic disease treated with bevacizumab monotherapy noted improvement in long-standing lymphedema. This pilot study was conducted to explore these observations prospectively. Methods: We used an existing biospecimen bank to conduct a case-control study to compare VEGF-A, -C, -D and VEGFR-3 serum concentrations in breast cancer pts with and without lymphedema (matched for age and LN status). In a separate pilot trial, pts with significant unilateral lymphedema receive bevacizumab, 15 mg/kg every 3 weeks. Baseline assessments include arm volume, interstitial fluid pressure (IFP), extracellular fluid volume by lymphometer (ECF), quality of life (QOL) and plasma VEGF-C, -D, and R3. IFP is measured serially for 24 hours after the first treatment; arm volume, ECF, QOL, and plasma VEGF-C/D/R3 are assessed at 3 and 6 weeks. Results: Samples were available for 16 pts with chronic lymphedema and 31 matched controls. Median VEGF-C levels were significantly increased in pts with lymphedema (6895 pg/ml vs. 5349 pg/ml, p=0.001). Twelve pts have been enrolled in the pilot trial. Median duration of lymphedema was 6.3 years (0.5–16.6) Median time since surgery was 5.5 years (1.9–17.6); median time since radiation (n=10) was 5.1 years (1.3–8.3). Complete IFP data is available in 9 pts. Baseline IFP was significantly higher in the affected compared to unaffected arm (7.63 vs. -0.87 mmHg; p=0.0023). Mean IFP in the affected arm decreased an average of 41.9% 24 hours after bevacizumab infusion (7.63 vs. 4.88 mmHg; p=0.07). ECF, VEGF-1, and VEGF-R3 levels all significantly decreased three weeks after initial treatment. Two patients met the definition of response with a ≥ 25% reduction in excess arm volume though the mean difference in excess arm volume did not significantly change (p=0.19). Conclusions: Preliminary data supports the hypothesis that VEGF plays a central role in the development and persistence of lymphedema after local therapy for breast cancer. Bevacizumab acutely decreases IFP, leading to a decrease in ECF and modest improvement in arm volume. [Table: see text]

Abstract #6142 Purpose: Lymphedema is a significant long-term complication of primary therapy for breast cancer but little is known regarding its etiology and persistence other than relation to the number of lymph nodes (LN) removed. We suggest the following model: Mechanical disruption of normal lymphatic drainage leads to increased back pressure on lymphatic vessels producing an increase in interstitial fluid pressure (IFP). The increased in IFP triggers local production of VEGF-C to stimulate compensatory lymphangiogenesis. As VEGF-A (and to a lesser extent –C) also increases vascular permeability, the consequent increase in interstitial fluid and edema predominate. Anecdotally, several patients (pts) with metastatic disease treated with bevacizumab monotherapy noted improvement in long-standing lymphedema. This pilot study was conducted to explore these observations prospectively. Methods: We used an existing biospecimen bank to conduct a case-control study to compare VEGF-A, -C, -D and VEGFR-3 serum concentrations in breast cancer pts with and without lymphedema (matched for age and LN status). In a separate pilot trial, pts with significant unilateral lymphedema receive bevacizumab, 15 mg/kg every 3 weeks. Baseline assessments include arm volume, interstitial fluid pressure (IFP), extracellular fluid volume by lymphometer (ECF), quality of life (QOL) and plasma VEGF-C, -D, and R-3. IFP is measured serially for 24 hours after the first treatment; arm volume, ECF, QOL, and plasma VEGF-C/D/R-3 are assessed at 3 and 6 weeks. Results: Samples were available for 16 pts with chronic lymphedema and 24 matched controls. Median VEGF-C levels were significantly increased in pts with lymphedema (6895 pg/ml vs. 5349 pg/ml, p=0.001). Median VEGF-A levels were slightly higher in pts with lymphedema (375 pg/ml vs.250 pg/ml, p=NS). Eight pts have been enrolled in the pilot trial thus far. Median duration of lymphedema was 4.4 years (2.2-16.6) Median time since surgery was 7.1 years (3.4-17.6); median time since radiation (n=7) was 4.4 years (2.3-7.9). Complete IFP data is available in 5 pts. Baseline IFP was significantly higher in the affected compared to unaffected arm (9.04 vs. -2.07 mmHg; p=0.0017). Median IFP in the affected arm decreased an average of 42.6% 24 hours after bevacizumab infusion (11.1 vs. 6.9 mmHg; p=0.09). Total arm circumference decreased by an average of 2.8 cm three weeks after initial treatment. Conclusions: Preliminary data supports the hypothesis that VEGF plays a central role in the development and persistence of lymphedema after local therapy for breast cancer. Inhibiting VEGF acutely decreases IFP and may be an effective treatment. Treatment and accrual to the pilot trial continues; full data will be available by December 2008. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6142.

Abstract #3121 Background: Lapatinib is a selective and highly potent dual, competitive inhibitor of erbB1 and erbB2 tyrosine kinases with clinical activity in erbB2-positive metastatic breast cancer (MBC). The combination of carboplatin, paclitaxel, and trastuzumab has been shown to have significant clinical activity in MBC as well as in the adjuvant setting. Given the synergy of dual inhibition with trastuzumab and lapatinib observed in both the preclinical and clinical settings, we assessed the feasibility, safety, and early clinical activity of paclitaxel, carboplatin, lapatinib with and without trastuzumab in patients (pts) with MBC. Methods: MBC pts previously untreated with trastuzumab or cytotoxic chemotherapy for metastatic or locally recurrent disease were enrolled into either Group (Grp) A (erbB2 positive) or B (erbB2 positive or negative). Escalating doses of lapatinib (planned range of 750-1500 mg/d) were administered in combination with (Grp A) or without (Grp B) trastuzumab (4 mg/kg followed by weekly 2 mg/kg infusions). Paclitaxel (80 mg/m2) and carboplatin (AUC 2 mg/ml*min) were administered on days 1, 8, 15 with cycles repeated every 28 days. Starting doses of lapatinib were 750 mg in Grp A and 1000 mg in Grp B. A standard 3 + 3 Phase I design is being used until the optimally tolerated regimen (OTR) dose level is determined. An additional 14 - 17 pts will be enrolled into each grp at the OTR dose level to further assess safety and tolerability. Results: Fourteen pts (Grp A n=8, Grp B n=6) have been enrolled in the study (median age 42 yrs, range 27-66). A median of 5.5 cycles (range 4-17) in Grp A and 3.5 (range 1-7) in Grp B have been administered. All pts had at least one adverse event (AE). In Grp A, grade 3 toxicities include neutropenia (50%), diarrhea (38%), rash (25%), vomiting (13%), hypokalemia (13%), and syncope (13%). No grade 4 toxicities were reported. In Grp B, grade 3 toxicities include fatigue (33%), hyponatremia (17%), menorrhagia (17%), dermatitis (17%), and rash (17%). One pt (in Grp B) had grade 4 neutropenia. No febrile neutropenia was observed. In Grp A, there was one DLT (Gr 3 diarrhea) at lapatinib 1000 mg. In Grp B, one DLT (Gr 4 neutropenia) at lapatinib 1000 mg has been observed. Clinical activity to date includes 8/8 objective responses in Grp A, (1 CR, 7 PR) and 2/6 objective responses (2 PR) in Grp B (all pts in Grp B were erbB2 negative). Dose escalation continues in both grps. Conclusions: Lapatinib may be administered safely in combination with carboplatin, paclitaxel, with and without trastuzumab at known effective doses for each. Clinical activity has been observed. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3121.

828 Background: Over 95% of breast cancers (BC) originate in the epithelial cells lining the breast duct. We piloted the use of the IDBM as a targeted drug delivery tool to infuse epi-containing NP into the breast duct. Methods: Epi, a naturally fluorescent anthracycline, was encapsulated into biodegradable NP (diameter < 1μ), which could then be suspended in saline for injection. Informed consent was obtained from women with planned mastectomies. In the operating room, the mastectomy was performed and the tumor specimen was removed for pathologic assessment. Via the IDBM, 3–5 ml of NP (200 mg/ml suspended in saline), containing 6% epi, were injected into a breast duct that had been previously marked with vital blue dye. The duct was dissected, frozen intact, and subsequently “step-sectioned”. Sections were examined by fluorescent microscopy to assess presence of intact NP within the ductal lumen. “Success” was defined as the presence of intact NP within the lumen of the distal one-third of the dissected ...