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Lei et al. J Cancer Metastasis Treat 2019;5:38 I http://dx.doi.org/10.20517/2394-4722.2019.12 Page 7 of 16

[37]
disease . One patient from this study acquired a tyrosine substitution to cysteine mutation (Y537C) at the
[37]
metastatic site, which was not detected prior to treatment . Taken together, these studies indicate the most
frequent ESR1 LBD point mutations are those affecting Y537 and D538 residues. Furthermore, the presence
of ESR1 point mutations predominately appear in late-stage breast cancer patients that have been treated
with multiple lines of endocrine therapies but rarely in treatment naïve cases. This strongly suggests a role
for ESR1 point mutations in acquired endocrine resistance and metastasis.

Although formalin-fixed paraffin-embedded tumor specimens are widely used for next generation
sequencing to capture ESR1 mutations used by studies as described above [37,53,54] , collection of plasma
circulating DNA to detect ESR1 mutations by droplet digital PCR (ddPCR) have now been implemented
in several clinical trials [56-59] . Such “liquid biopsies” have shown that collecting circulating DNA samples
maintains the genomic landscape of the primary tumor suggesting that less invasive detection methods
may efficiently identify ESR1 point mutations once the disease has become resistant to treatment and/or has
become metastatic. Interestingly, Y537 and D538 substitutions were identified in 7% of ER+ primary tumors
using ddPCR, which may lead us to review the conclusion that ESR1 point mutations rarely exist in primary
tumor, towards the idea that rare ESR1 mutant sub-clones exist in primary breast tumors that become
selected for over time .
[60]
Experimental models of ESR1 point mutations
Several preclinical breast cancer models harboring ESR1 LBD point mutations have been generated,
providing research platforms to characterize the functional, transcriptional, and pharmacological properties
of these mutations. ER point mutant proteins have been overexpressed by transfecting [37,53,54] or transducing
lentiviral vectors [55,61] encoding ESR1 mutant constructs into various ER+ breast cancer cell line models.
The growth promoting properties of ESR1 mutant expressing cell line models have shown that ESR1
LBD mutants drive hormone-independent proliferation that is resistant to tamoxifen treatment [23,37,47,53,54] .
Although fulvestrant efficiently inhibited the growth of point mutation bearing cells in a dose-dependent
manner, growth was not reversed to levels of wild-type ESR1 expressing cells [37,47] .

Since the expression of exogenous ESR1 variant transcripts encoded by expression vectors is often initiated
from non-endogenous human promoters that drive very high expression of constructs, it is unlikely to
mimic the expression levels in human breast tumors harboring ESR1 point mutations. To more accurately
recapitulate tumor-related ESR1 mutational events, CRISPR/Cas9 approaches have been utilized to knock
in ESR1 mutated sequences into ER+ breast cancer cells [62,63] . Both heterozygous and homozygous knock-in
models have been shown to mediate resistance to endocrine therapies [62,63] .

Transcriptional properties of ESR1 mutations in the LBD include their ability to drive constitutive hormone-
independent transcriptional activation and enhance cell proliferation [23,37,47,53-55] . Human embryonic kidney
293T cells transfected with Y537C, Y537N, and D538G mutant constructs strongly activate an ERE-luciferase
reporter in a ligand-independent manner compared to wild-type ER. Luciferase activity was unaffected by
clinically relevant doses of tamoxifen and fulvestrant, however, high doses of these agents blocked ESR1
mutant driven ERE-luciferase reporter activity [37,53-55] . These ESR1 point mutations have also been shown to
drive estrogen-independent activation of ER target genes in ER+ breast cancer cells [37,53,54] . The recruitment of
ESR1-Y537S mutant to ER target genes and their expression driven by the mutant were further validated by
[62]
ChIP-seq and RNA-seq .

ESR1 mutant-driven estrogen-independent tumor growth was also validated in both ER+ cell xenografts and
patient-derived xenograft (PDX) models [47,53] . A PDX harboring ESR1-Y537S, WHIM20, has been generated
from a patient with endocrine-refractory metastatic ER+ breast cancer that retains genomic features of the
[47]
human counterpart . This WHIM20 PDX model demonstrated estrogen-independent tumor growth .
[47]