Erratum to: Humanization of the mouse mammary gland by replacement of the luminal layer with genetically engineered preneoplastic human cells.

Due to a typesetting error, the labelling was changed and the figures in this article [1] were presented in the order 2, 4, 10, 6, 1, 3, 5, 7, 8, 9, 11, 12, 13, 14 and the supplementary figure links were inverted. The revised version has the figures in the correct order.

Humanization of the mouse mammary gland by replacement of the luminal layer with genetically engineered preneoplastic human cells.

INTRODUCTION: The cell of origin for estrogen receptor α-positive (ERα+) breast cancer is probably a luminal stem cell in the terminal duct lobular units. To model these cells, we have used the murine myoepithelial layer in the mouse mammary ducts as a scaffold upon which to build a human luminal layer. To prevent squamous metaplasia, a common artifact in genetically-engineered breast cancer models, we sought to limit activation of the epidermal growth factor receptor (EGFR) during in vitro cell culture before grafting the cells. METHODS: Human reduction mammoplasty cells were grown in vitro in WIT medium. Epidermal growth factor in the medium was replaced with amphiregulin and neuregulin to decrease activation of EGFR and increase activation of EGFR homologs 3 and 4 (ERBB3 and ERBB4). Lentiviral vectors were used to express oncogenic transgenes and fluorescent proteins. Human mammary epithelial cells were mixed with irradiated mouse fibroblasts and Matrigel, then injected through the nipple into the mammary ducts of immunodeficient mice. Engrafted cells were visualized by stereomicroscopy for fluorescent proteins and characterized by histology and immunohistochemistry. RESULTS: Growth of normal mammary epithelial cells in conditions favoring ERBB3/4 signaling prevented squamous metaplasia in vitro. Normal human cells were quickly lost after intraductal injection, but cells infected with lentiviruses expressing CCND1, MYC, TERT, BMI1 and a short-hairpin RNA targeting TP53 were able to engraft and progressively replace the luminal layer in the mouse mammary ducts, resulting in the formation of an extensive network of humanized ducts. Despite expressing multiple oncogenes, the human cells formed a morphologically normal luminal layer. Expression of a single additional oncogene, PIK3CA-H1047R, converted the cells into invasive cancer cells. The resulting tumors were ERα+, Ki67+ luminal B adenocarcinomas that were resistant to treatment with fulvestrant. CONCLUSIONS: Injection of preneoplastic human mammary epithelial cells into the mammary ducts of immunodeficient mice leads to replacement of the murine luminal layer with morphologically normal human cells. Genetic manipulation of the injected cells makes it possible to study defined steps in the transformation of human mammary epithelial cells in a more physiological environment than has hitherto been possible.

Validation of a yeast functional assay for p53 mutations using clonal sequencing.

We have previously tested biopsies from 1469 breast tumours with a p53 functional assay in the context of a prospective clinical trial (EORTC 10994/BIG 1-00). The goal of the trial was to determine whether p53 status could be used to select patients who would benefit from inclusion of taxanes in anthracycline-based chemotherapy. The results of the trial were negative. To test whether this was because the functional assay misclassified the tumours, we have reanalysed two groups of biopsies by Sanger sequencing and Roche 454 next generation sequencing (NGS). Comparison of yeast data with pooled cDNA sequencing data in an initial cohort of 69 biopsies showed that conventional sequencing is insensitive when the mutant p53 content is low. A second cohort of 48 biopsies was used to compare directly the yeast assay with Sanger and NGS technology. The mutant sequence was difficult to detect in sequence chromatograms of pooled cDNA, whereas NGS unequivocally identified mutations in every case classified as mutant by the functional assay. The NGS data showed that small deletions, probably caused by PCR splicing, account for most of the unexplained background in the yeast assay. We conclude that mutation detection techniques that test multiple clones, such as the p53 functional assay and NGS, are more reliable than Sanger sequencing of pooled DNA; that the high p53 mutation rate (44%) seen with the yeast assay in the EORTC 10994/BIG 1-00 trial reflects this high sensitivity; and that NGS with Roche 454 technology could be used to identify the p53 mutations in the remaining tumours previously tested in yeast in the EORTC10994/BIG 1-00 trial.