Abstract Spontaneous immortalization of benign mammary epithelial cells (MEC) in culture is very rare. Models of MEC immortalization suggest that cells escape M0 growth arrest (selection) by silencing p16 expression after which they encounter a second growth arrest, M1 (agonescence), characterized by telomere shortening and profound genomic instability. Pre-selection, post-selection, and spontaneously immortalized MEC from a woman with a germline STK11 mutation were subjected to a comprehensive “OMICS” evaluation including miRNA, transcriptome, methylome, exome, and comparative genomic hybridization (CGH) in order to understand the molecular events associated with MEC immortalization. Principle component analysis of gene expression identified 4 significant components, the first of which showed the greatest separation between the different time points. The most notable expression changes were upregulation of FOXQ1, IL1-related genes, Ras-related genes, and fibronectin and downregulation of COL1, COL6, CK19, and CDH1. MEC gradually acquired global hypomethylation of non-CpG DNA and regional hypermethylation of CpG islands (including p16). A classifier based on methylation pattern showed that immortalized cells moved away from identity with The Cancer Genome Atlas (TCGA) benign samples towards TCGA breast cancer samples. Immortalization was associated with a modest accumulation of mutations and a modestly greater number of DNA copy number alterations than was observed in earlier passage cells but no evidence of general genomic instability. Immortal cells showed a large deletion at 10q13-15 and deletions at 10p11-15 and 17p11-13 that were associated with reduced expression of genes in those regions. Post-selection bulk cell populations are quite distinct from early passage cells, but are not direct precursors of the immortal cells as some DNA copy number alterations acquired by post-selection cells were not observed in the immortalized cells. Rather, in this instance, it is likely that post-selection cells established conditions conducive to the immortalization of a rare cell in the mix. This has implications for the development of tissue-based approaches for breast cancer risk stratification. Citation Format: David Euhus, Dawei Bu, Michael Considine, Leslie Cope. Spontaneous immortalization of human mammary epithelial cells from a woman with a germline STK11 mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 843.