Identification of altered growth phenotypes in human breast cancer cells using cell culture methods that support growth of normal and neoplastic mammary epithelial cells

Over the past several years our laboratory has been studying factors that regulate proliferation of normal human mammary epithelial (HME) cells in order to better understand the alterations in cellular growth control mechanisms that occur during breast cancer development. To perform these experiments, we have either modified or developed cell culture methods for the isolation and growth of normal and neoplastic HME cells obtained from patient biopsy specimens. From these studies we have found that normal HME cells of the luminal lineage (the lineage from which breast cancer arises) have strict requirements for specific growth factor combinations for in vitro growth. Furthermore, these cells have a finite proliferative lifespan in culture. By contrast, human breast cancer (HBC) cells isolated from primary and metastatic sites exhibit many growth phenotypes that distinguish them from normal cells. First, whereas normal HME cells proliferate in culture with doubling times of 24–36 hours, HBC cells obtained from patient samples proliferate with doubling times of 100–200 hours. These proliferation kinetics are consistent with the rate at which these cells proliferate in vivo . This observation indicates that there are fundamental differences in growth regulation between normal and neoplastic mammary epithelial cells. Second, the majority of HBC cells isolated from human samples exhibit an extended proliferative lifespan in culture. Whereas normal HME cells undergo cell senescence after 15–20 population doublings, HBC cells often give rise to cell lines with indefinite proliferative potential. Third, HBC cells become independent of growth factors which are strictly required by normal HME cells for growth under defined conditions. In our experiments, escape from the requirements of exogenous epidermal growth factor (EGF) has been observed in cells from four patient-derived samples. Interestingly, the cellular mechanisms by which cells become EGF-independent for growth is different in cell lines isolated from different patients. Two breast cancer cell lines isolated in our laboratory proliferate continuously in serum-free, EGF-free medium and do not express EGF receptors. Thus, these cells are completely independent of EGF-mediated signalling pathways for their growth. A third cell line isolated in our laboratory has an amplified EGF receptor gene and overexpresses EGF receptor protein. Western blot analysis indicates that the tyrosine residues of the EGF receptor proteins in these cells are highly phosphorylated. These cells do not secrete any EGF-like growth factors that could be activating the receptors in an autocrine manner. This suggests that amplification and overexpression of EGF receptors can yield constitutively activated receptors that provide a mitogenic signal in the absence of a stimulatory ligand. Finally, we have analyzed the EGF requirements of human breast cancer cells that overexpress the erb B-2 receptor as a result of gene amplification. The results of these experiments indicated that overexpression of erb B-2 is, by itself, insufficient to overcome the EGF requirements of human breast cancer cells. However, one cell line that has a 15- to 20-fold amplification of erb B-2 and which expresses very high levels of tyrosine phosphorylated erb B-2 protein, is EGF-independent for growth. Thus, cell culture systems that allow proliferation of normal HME cells and HBC cells under well-defined culture conditions can result in identification of altered growth phenotypes associated with the neoplastic progression of breast cancer cells. In addition, isolation of cells exhibiting altered growth phenotypes may lead to insights as to the genetic mechanisms resulting in altered growth regulation in breast cancer cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38456/1/240531151_ftp.pd

Hypofractionated postmastectomy radiation therapy is safe and effective: First Results from a prospective phase II trial

© 2017 by American Society of Clinical Oncology. Purpose: Conventionally fractionated postmastectomy radiation therapy (PMRT) takes approximately 5 to 6 weeks. Data supporting hypofractionated PMRT is limited. We prospectively evaluated a short course of hypofractionated PMRT, in which therapy was completed in 15 treatment days. Patients and Methods: We delivered PMRT at a dose of 36.63 Gy in 11 fractions of 3.33 Gy over 11 days to the chest wall and the draining regional lymph nodes, followed by an optional mastectomy scar boost of four fractions of 3.33 Gy. Our primary end point was freedom from any grade 3 or higher toxicities. We incorporated early stopping criteria on the basis of predefined toxicity thresholds. Results: We enrolled 69 women with stage II to IIIa breast cancer, of whom 67 were eligible for analysis. After a median follow-up of 32 months, there were no grade 3 toxicities. There were 29 reported grade 2 toxicities, with grade 2 skin toxicities being the most frequent (16 of 67; 24%). There were two patients with isolated ipsilateral chest wall tumor recurrences (2 of 67; crude rate, 3%). Threeyear estimated local recurrence-free survival was 89.2% (95% CI, 0.748 to 0.956). The 3-year estimated distant recurrence-free survival was 90.3% (95% CI, 0.797 to 0.956). Forty-one patients had chest wall reconstructions; three had expanders removed for infection before radiation therapy. The total rate of implant loss or failure was 24% (9 of 38), and the unplanned surgical correction rate was 8% (3 of 38), for a total complication rate of 32%. Conclusion: To our knowledge, our phase II prospective study offers one of the shortest courses of PMRT reported, delivered in 11 fractions to the chest wall and nodes and 15 fractions inclusive of a boost. We demonstrated low toxicity and high local control with this schedule. On the basis of our data, we have designed a cooperative group phase III prospective, randomized trial of conventional versus hypofractionated PMRT that will activate soon