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Page 4 of 16 Lei et al. J Cancer Metastasis Treat 2019;5:38 I http://dx.doi.org/10.20517/2394-4722.2019.12

Dysregulation of cell cycle components is common in ER+ breast cancer, particularly the Cyclin D-CDK4/6-
[19]
Rb axis in the luminal B subtype . This includes amplification of Cyclin D1 (CCND1), gene copy gain
[19]
of CDK4 and loss of negative regulators such as p16 and p18 (CDKN2A and CDKN2C) . Together with
downstream activity from tyrosine kinase growth factor signaling described earlier, these events promote
[20]
phosphorylation of Rb and resistance to endocrine therapy . CDK4/6 inhibitors such as palbociclib and
ribociclib, are now FDA approved for use in combination with endocrine therapy to treat advanced stage ER+
disease. Other studies are now examining the use of such inhibitors to treat early stage ER+ disease in both
neoadjuvant and adjuvant settings (ClinicalTrials.gov identifiers for PALLET NCT02296801 and PALLAS
[21]
NCT02513394, respectively). Some trials have already reported promising results in the neoadjuvant setting .

In addition to metastatic breast tumors expressing wild-type ER [Figure 1A], alterations in ESR1 itself, such
[22]
as ESR1 amplifications have been identified in metastatic ER+ disease [Figure 1B]. Other ESR1 alterations
found in endocrine therapy resistant breast tumors include point mutations in the ligand-binding domain
[23]
(LBD) [Figure 1C] that confer constitutive hormone-independent activation of ER and are now a well-
[24]
described mutational mechanism identified in up to 40% of metastatic breast cancer cases . These are
[25]
especially enriched in tumors pretreated with aromatase inhibitors . Emerging evidence now suggests that
chromosomal rearrangement events involving ESR1 are yet another ESR1 mutational mechanism driving
endocrine therapy resistance and metastatic disease progression [Figure 1D]. Hereon, we focus on the
spectrum of ESR1 aberrations underlying treatment resistance and metastasis in ER+ breast cancer.

ESR1 AMPLIFICATION
The copy number increase of a confined area of a chromosome is defined as gene amplification/gain [Figure 1B]
which may result in protein overexpression of the amplified gene therefore driving tumor biology. For
[27]
[26]
example, ERBB2 amplification and fibroblast growth factor receptor 1 gene (FGFR1) amplification
are drivers of therapeutic resistance and poor prognosis in ER+ breast cancer. The discovery of ESR1 gene
[28]
amplifications in 1990 sparked intense interest in investigating the role of this mutational event to be a
potential driver of endocrine therapy resistance and recurrent disease in ER+ breast tumors.

Incidence of ESR1 amplifications in ER+ breast cancer
ESR1 amplification is found in up to 30% of ER+ breast tumors [22,28-37] depending on the detection method
[29]
[38]
and scoring systems . A study by Holst et al. that analyzed over 2,000 breast tumors, showed that 20.6%
of tumors harbored ESR1 amplifications and 14% showed ESR1 copy number gain by using fluorescence in
[29]
situ hybridization (FISH) method and validated by quantitative PCR . Nearly all ESR1 amplified tumors
in these samples also expressed high levels of ER protein by immunohistochemistry. Additional analysis
from precancerous ductal and lobular carcinoma in situ (DCIS and LCIS) breast tumors showed over one-
third of these samples also harbored ESR1 amplifications suggesting that ESR1 amplifications present in
early-stage breast cancer may drive disease progression. Two other independent studies that also used FISH,
[29]
both showed that ESR1 amplification frequency is between 20%-22% [34-35] , consistent with Holst et al. . In
[30]
[31]
[33]
[32]
contrast, other studies by Brown et al. , Horlings et al. , Reis-Filho et al. , and Vincent-Salomon et al. ,
have shown a much lower frequency of ESR1 amplifications, in which ESR1 amplification or gain was less
than 5% by using array comparative genomic hybridization (aCGH) and validated by FISH by the majority
of these studies. Another study which used a multiplex ligation-dependent probe amplification (MLPA)
approach to analyze 104 invasive breast cancers identified 16% of samples harbored ESR1 amplifications
[36]
consisting of low level gains . A variation in the frequency of ESR1 amplification found among metastatic
[37]
breast samples has also been reported. A seminal study from Jeselsohn et al. examined ESR1 amplification
in the metastatic setting using next generation sequencing approaches. They reported the frequency of ESR1
[37]
amplification in ER+ tumors at less than 2% in both the primary and metastatic setting . Using NanoString
sequencing approaches, a recent study reported that 13% of ER+ metastatic breast tumors harbored ESR1
amplifications. Interestingly, the authors found an enrichment of ESR1 amplifications in bone metastatic