Introduction: The lack of estrogen receptor (ER) expression is the primary cause of de novo resistance of breast cancers to endocrine therapy. In contrast, in most cases of acquired endocrine resistance, ER is expressed and other mechanisms of resistance have been proposed, such as ER mutations. Pre-clinical studies demonstrated a small number of specific point mutations that can enhance ER function. However, the studies on clinical samples performed in the 1990's were limited by small sample size, lack of detailed clinical correlation and lacked the sensitivity of next-generation sequencing (NGS). Therefore, in this study we sought to comprehensively investigate the frequency and functional significance of ER mutations throughout the progression of breast cancer from primary disease to advanced metastatic disease using targeted NGS.

Methods: In this retrospective study, a total of 249 tumor specimens were analyzed. The specimens include 134 ER positive and, as controls, 115 estrogen receptor negative tumors. The estrogen receptor positive samples consist of 58 primary breast cancers and 76 metastatic sample. All tumors were sequenced with high coverage using NGS targeting the coding sequence of ER and an additional 181 cancer-related genes.

Results: Recurring somatic mutations at codons 537 and 538 within the ligand-binding domain of the estrogen receptor were detected in ER positive metastatic tumors. Overall, the frequency of these mutations was 12% (95% CI 6%-21%) in metastatic patients compared with none in the primary cases. In total there were 9 recurring somatic mutations; Y537C (11%), Y537N (33%), Y537S (22%) and D538G (33%). In addition in a small number of paired primary and metastatic samples from the same patient, these mutations were found only in the metastatic specimens. In a subset of heavily pre-treated patients the frequency was 20% (5/25, 95% CI 7%-41%). ER activating mutations were not detected in any stage of ER negative disease. ER alterations were not mutually exclusive with any of the other commonly altered genes and of the most frequently altered genes, all but ER alterations displayed similar frequencies across primary and metastatic specimens. Functional studies in cell line models demonstrated that these ER mutations render ER constitutively active and confer resistance to hormone deprivation, tamoxifen and fulvestrant.

Conclusions: Herein, we reveal functional ER mutations as potential drivers of endocrine resistance during the progression of ER positive breast cancer. The absence of detectable mutations in the primary tumors suggests clonal evolution as the mechanism of resistance. Thus, these mutations have the potential to be an important genetic biomarker of endocrine resistance in ER positive metastatic breast cancer and could assist in clinical decision making as disease progresses. Our findings also underscore the value of repeated biopsies of metastatic lesions. Lastly, since the frequencies of these mutations are substantial when sensitive testing methods are used in the correct clinical context, pre-clinical and clinical studies to identify novel therapeutics that can overcome this resistance are warranted.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S3-06.