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are partially sensitive to fulvestrant, requiring higher doses of fulvestrant compared to controls [37,47,63] .
Moreover, fulvestrant did not completely block transcriptional activity nor cell proliferation compared to
control cells expressing wild-type ESR1. Of note, ESR1 mutants showed differential responses to fulvestrant.
Y537S required the highest dose to completely block transcriptional activity and cell proliferation compared
[63]
to other mutants, D538G, E380Q and S463P . Using an MCF7 xenograft model, ESR1 mutants also showed
differential responses to fulvestrant. Tumor growth of E380Q, S463P and D538G expressing tumors were
[63]
significantly reduced while Y537S tumors showed resistance to treatment . Given the inconvenience and
poor bioavailability of intramuscular fulvestrant injections, second-generation SERDs, such AZD9496, that
can be orally administrated have been tested and showed anti-proliferative ability in endocrine resistant
experimental models cell xenograft models [63,68] . AZD9496 which has improved bioavailability compared to
fulvestrant, was able to provide greater suppression of tumor growth in the Y537S MCF7 xenograft model
[63]
and in a D538G PDX model compared to fulvestrant treatment . A phase I clinical trial with AZD9496
in extensively pretreated advanced ER+ breast cancer patients has recently been completed with promising
[69]
results, providing disease stabilization to the study cohort . These results suggest that newer generation
SERDs with improved bioavailability could be an attractive therapeutic option to treat endocrine-refractory
breast tumors driven by ESR1 mutations.

Treatment of late-stage ER+ breast cancer patients with CDK4/6 inhibitors in combination with endocrine
therapy has been tremendously successful. CDK4/6 inhibitors have also been tested in PDX breast
[70]
cancer models harboring ESR1 point mutations. Wardell et al. reported the suppressive effects of a
CDK4/6 inhibitor, palbociclib, on endocrine-refractory PDX tumors as long as the downstream target
retinoblastoma (Rb) protein was expressed. Used as monotherapy or in combination with a hybrid SERM/
SERD, bazedoxifene, palbociclib suppressed tumor growth of a WHIM20 PDX tumor harboring an ESR1-
Y537S mutant. In contrast, palbociclib was ineffective in inhibiting the growth of WHIM43, a PDX naturally
bearing ESR1-D538G mutant due to the lack of Rb protein expression, suggesting that Rb is a determinant
of CDK4/6 treatment response. CDK4/6 inhibitors also showed favorable therapeutic effects in treatment-
[59]
resistant ER+ patients harboring ESR1 point mutations .

Currently, screening of ESR1 point mutations have not been used as biomarkers to predict response to
therapy in the clinic. Wild-type ER, human epidermal growth factor receptor 2 (HER2), and progesterone
receptor (PR), are histopathological markers that guide therapeutic selection. In clinical management of
metastatic ER+ breast cancer, SERDs, such as fulvestrant is used for patients with resistance to AIs and
tamoxifen without regard for ESR1 mutation status. An analysis of BOLERO-2, a phase III clinical trial
that enrolled ER+ breast cancer patients with locally advanced or metastatic disease whom progressed on
AI, evaluated the prevalence of the two most frequent ESR1 point mutations, Y537S and D538G and their
[56]
effects on patient outcomes in ER+ metastatic patients . Having either one or two of these mutations was
associated with decreased overall survival. In the PALOMA-3 clinical trial which enrolled ER+ breast cancer
patients with advanced, endocrine refractory disease, palbociclib combined with fulvestrant led to longer
PFS than fulvestrant alone [59,71] . 69% of patients from the PALOMA-3 were analyzed for ESR1 mutation
status, which showed that 25% of these cases harbored ESR1 mutations consisting mainly of Y537S, Y537N,
[59]
D538G, and E380Q mutations . However, palbociclib was found to provide equal benefit regardless of
ESR1 mutation status. Although these studies indicate that the presence of ESR1 mutations may predict poor
outcomes, they also highlight the need for more analyses of studies investigating the predictive value of ESR1
mutation status and response to therapy once the disease has become endocrine therapy resistant.

The development of sequencing technologies and the various models to recapitulate ESR1 mutant bearing
tumors allow insightful studies into the landscape and targeted therapies of activating point mutations in
the ESR1 LBD. Further studies are needed to address the use of ESR1 mutations as predictive biomarkers to
stratify patient subsets and predict ESR1 mutation specific therapeutic vulnerabilities.