Ratsch, S. B., Gao, Q., Srinivasan, S., Wazer, D. E. and Band, V. Multiple Genetic Changes Are Required for Efficient Immortalization of Different Subtypes of Normal Human Mammary Epithelial Cells.Breast cancer is the second leading cause of cancer-related deaths of women in the U.S. About 180,000 new cases of breast cancer are diagnosed each year, a quarter of them fatal. Early detection is the key to the survival of these patients. However, there are no molecular markers to detect breast cancer at very early stages. A hurdle in understanding the early molecular changes in breast cancer has been the difficulty in establishing premalignant lesions and primary breast tumors as in vitro cell cultures. Normal epithelial cells grow for a finite life span and then senesce. Immortalization is defined by continuous growth of otherwise senescing cells and is believed to represent an early stage in tumor progression. To examine these early stages, we and others have developed in vitro models of mammary epithelial cell immortalization. These models have been extremely important in understanding the role of various tumor suppressor pathways that maintain the normal phenotypes of mammary epithelial cells. In this paper, we describe the establishment of these models and their relevance to understanding the molecular changes that occur in early breast cancer. These models have helped to identify molecular changes that occur in early breast cancers and appear to be well suited to identify novel markers for early diagnosis of breast cancer.