B-S Herbert

BACKGROUND: Metastatic tumour cells are characterised by acquisition of migratory and invasive properties; properties shared by cells, which have undergone epithelial-to-mesenchymal transition (EMT). Disabled-2 (Dab2) is a putative tumour suppressor whose expression has been shown to be downregulated in various cancer types including breast cancer; however, its exact function in suppressing tumour initiation or progression is unclear. METHODS: Disabled-2 isoform expression was determined by RT-PCR analysis in human normal and breast tumour samples. Using shRNA-mediated technology, Dab2 was stably downregulated in two cell model systems representing nontumourigenic human mammary epithelial cells. These cells were characterised for expression of EMT markers by RT-PCR and western blot analysis. RESULTS: Decreased expression of the p96 and p67 isoforms of Dab2 is observed in human breast tumour samples in comparison to normal human breast tissue. Decreased Dab2 expression in normal mammary epithelial cells leads to the appearance of a constitutive EMT phenotype. Disabled-2 downregulation leads to increased Ras/MAPK signalling, which facilitates the establishment of an autocrine transforming growth factor β (TGFβ) signalling loop, concomitant with increased expression of the TGFβ2 isoform. CONCLUSION: Loss of Dab2 expression, commonly observed in breast cancer, may facilitate TGFβ-stimulated EMT, and therefore increase the propensity for metastasis.

Abstract Background: Preclinical studies have shown the combination of imetelstat (GRN163L), an inhibitor of telomerase, and T results in synergistic growth inhibition and restoration of T sensitivity in T-resistant cells (Clin Can Res 12(10):3184–92, 2006). Here we translate those findings to the clinic with the first-in-man phase I trial of imetelstat+ T in patients (pts) with T-refractory HER2+ metastatic disease. Methods: T (6 mg/kg q3 wk) was administered with increasing doses of imetelstat (240/300/375 mg/m2 q3 wk) using a standard 3+3 dose escalation design. Maximum Tolerated Dose (MTD) was based on toxicity observed during cycle 1. Responding or stable pts continued treatment until progression. Tumor biopsy and bone marrow aspirate for biologic correlates were obtained at baseline and prior to cycle 2. Limited pharmacokinetics (PK) for T and imetelstat were included. Results: Ten pts were enrolled; median age was 54 (28–64). Patients were extensively pre-treated with the number of prior regimens ranging from 3 to >12. Prior cytotoxic therapies included anthracyclines (n = 9), taxanes (n = 9), vinorelbine (n = 8), capecitabine (n = 8), gemcitabine (n = 6), ixabepilone (n = 4), and eribulin (n = 4). Prior anti-HER2 targeted therapies included trastuzumab (n = 10), capecitabine (n = 8), T-DM1 (n = 6), and pertuzumab (n = 1). Therapy was well tolerated; no treatment related grade 4 toxicities were observed. Myelosuppression was limited to one pt with Grade 2 anemia and one each with Grade 2/3 thrombocytopenia. MTD was not reached. There were no objective responses; 2 pts. in cohort 3 had SD. Serial tumor biopsies and bone marrow aspirates have been analyzed for 7 pts (cohort 1: 001–004; cohort 2: 005–007; cohort 3: analysis ongoing). Tumor hTERT (p = ns) and phosphorylated HER2 (p = 0.03) decreased after treatment in nearly all pts. Bone marrow S-phase did not change consistently with treatment. Conclusion: The combination of trastuzumab + imetelstat is well tolerated and results in decreases in HER2 phosphorylation similar to that seen in preclinical models. Additional biologic correlates and PK analyses are ongoing. Further study of this combination in less heavily pre-treated patients is planned. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-18-13.

Abstract 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.

Abstract Background: BRCA1 is a tumor suppressor gene with a variety of functions related to safeguarding genomic integrity. The regulation of telomere length is also crucial in maintaining genomic stability, with critically short telomeres leading to telomere uncapping, end-to-end fusions, activation of the DNA damage response, and cell cycle arrest. The objective of this study was to determine whether GRN163L, a telomerase template antagonist currently in clinical trials, has enhanced activity in BRCA1 mutant breast or ovarian cancer cell lines compared to BRCA1 wild-type breast/ovarian cancer cell lines. Methods: We utilized a panel of breast and ovarian cancer cell lines harboring both N-terminal and C-terminal BRCA1 mutations. This cell line panel included two isogenic cell line pairs reconstituted with wild-type BRCA1 (BRCA1 mutant: HCC1937 and UWB1.289; BRCA1 wild-type reconstituted: HCC1937+BRCA1 and UWB1.289+BRCA1). We assessed the effects of GRN163L or a mismatch oligonucleotide (MM) on telomerase activity and telomere length using the TRAP (Telomere Repeat Amplification Protocol) and TeloTAGGG (Roche) assays, respectively. Clonogenic survival assays, proliferation assays, and immunofluorescence were used to assess cellular responses to GRN163L and their molecular mechanisms. Results: All cell lines tested exhibited a dose-dependent response to treatment with GRN163L, but not a mismatch control oligonucleotide. The majority of the cell lines tested showed gross morphological changes as early as 12 hours following inhibitor treatment. Progressive telomere shortening was evident after 3-week treatment with GRN163L, but not after treatment with MM. We observed differential sensitivity between BRCA1 mutant and wild-type isogenic cell line pairs to GRN163L in clonogenic survival assays. GRN163L pretreatment was synergistic with cisplatin in UWB1.289 and UWB1.289+BRCA1 cells. Simultaneous treatment with GRN163L and doxorubicin was synergistic in HCC1937 and HCC1937+BRCA1 cells. Long-term (12-week) treatment with GRN163L preferentially induced complete cell death in HCC1937 (BRCA1 mutant) breast cancer cells compared to HCC1937 cells reconstituted with wild-type BRCA1. One-week treatment with GRN163L was sufficient to induce γ-H2AX foci, with more γ-H2AX positive cells in the UWB1.289 BRCA1 mutant cell line. Conclusions: Telomerase inhibition may be a viable treatment in BRCA1 mutant breast or ovarian cancers. These data provide insights into further investigations on the role of BRCA1 in the DNA damage response to GRN163L or combination treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-08-01.