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Page 6 of 16 Lei et al. J Cancer Metastasis Treat 2019;5:38 I http://dx.doi.org/10.20517/2394-4722.2019.12

mechanism . This study also revealed that both CYP19A1 and ESR1 were frequently co-amplified in AI
[51]
treated patients, further suggesting that these two amplification events may function collaboratively. To
better understand the role of CYP19A1 amplification and endocrine therapy resistance, a LTED MCF7 ER+
breast cancer cell model was used which was found to acquire copy number alterations around the CYP19A1
[51]
locus compared to parental cells MCF7 cells . Elevated levels of both CYP19A1 mRNA and CYP19A1
protein were observed in CYP19A1 amplified LTED cells compared to parental cells. The functional
consequences of CYP19A1 amplification in the LTED cells were increased aromatase activity, enhanced
ER recruitment to regulatory regions on DNA of target genes and their transcriptional activation leading
[51]
to reduced sensitivity to AI treatment . These results suggest that CYP19A1 amplification, in addition to
ESR1 amplification, could potentially represent biomarkers of endocrine therapy resistance. More studies
are needed to validate these findings in more patient datasets. Furthermore, deeper studies focusing on how
these amplification events contribute to the metastatic behavior of endocrine-refractory ER+ breast tumors
are needed. These results highlight the possibility that response to standard-of-care endocrine therapies are
not only as a consequence of ESR1 amplification but may also be critically dependent on the status of the
target genes of endocrine therapies themselves.

ESR1 POINT MUTATIONS
When patients with ER+ breast cancer relapse, up to 15% have lost ER expression and therefore targeting ER
in this population is likely to be ineffective, although false negative ER results are a concern if the ER analysis
was conducted on bone biopsies exposed to acid formalin, or if the analysis was conducted on samples prone
to degradation such as cells detected in pleural fluid. The remaining 85% of patients may initially benefit
from first-line endocrine therapy, but metastatic disease progression due to acquired resistance is inevitable.
One well-established mechanism explaining this relentless pattern of acquired endocrine therapy resistance
is the acquisition of activating point mutations that cluster within the ligand-binding domain (LBD) of
ESR1 [Figure 1C]. Substitution of tyrosine at position 537 to serine (Y537S) in the LBD of ESR1 was first
[52]
reported to confer constitutive, ligand-independent activity of ER in experimental breast cancer models .
However, such mutations were not known to occur in human tumors until Fuqua et al. reported that
[23]
estrogen-independent activation could be driven by another Y537 substitution, Y537N, that was identified in
a metastatic sample from a breast cancer patient who experienced disease progression on hormonal therapy.
This study also showed that Y537N was able to drive resistance to tamoxifen in experimental models.

Frequent ESR1 point mutations in endocrine-refractory, metastatic ER+ breast cancer
Advances in sequencing technologies have allowed more sensitive detection and thus insights into the
landscape of ESR1 LBD point mutations in both primary and metastatic ER+ breast tumors. Three ESR1
mutations, Y537S, Y537N, and D538G were identified by next-generation sequencing in 14 out of 80 patient
[53]
samples with endocrine-refractory, metastatic ER+ breast cancer . Notably, all breast tumors from patients
that were found to harbor ESR1 LBD point mutations were treated with AIs. Interestingly, these alterations
were not detected in matched primary samples and were also not detected in separate large sets of treatment
naïve patients. Analysis of an independent ER negative (ER-) cohort also failed to detect any ESR1 point
[53]
mutations in the LBD . Although ESR1 mutations were found in 3% of primary samples in this population,
alterations in Y537 and D538 residues of ESR1 were enriched in patients treated extensively with AIs .
[53]
These results suggest that these ESR1 LBD mutations are acquired, or detected, in patients after treatment
with endocrine therapy.

In addition to Y537 alterations, frequent amino acid substitution of aspartate 538 to glutamate (D538G)
[54]
was identified in liver metastases from 5 out of 13 metastatic ER+ breast samples . Another study which
enrolled 11 metastatic ER+ breast cancer patients with exposure to serial endocrine therapies, identified that
over half of these patient’s metastatic samples harbored ESR1 mutations localized in the LBD, that included
Y537S, Y537C, Y537N, D538G, and L536Q mutations . Further evidence for the recurrent presence of Y537
[55]
and D538 mutations in the LBD of ESR1 was shown in 9 out of 76 metastatic samples from patients with ER+