Published OnlineFirst July 5, 2017; DOI: 10.1158/1078-0432.CCR-17-1173

ClinicalCancerResearch

Personalized Medicine and Imaging

Detection of Activating Estrogen Receptor
Gene (ESR1) Mutations in Single Circulating
Tumor Cells
Carmela Paolillo1,2, Zhaomei Mu3, Giovanna Rossi3, Matthew J. Schiewer1,
Thomas Nguyen1, Laura Austin1, Ettore Capoluongo2, Karen Knudsen1,
Massimo Cristofanilli3, and Paolo Fortina1,4

Abstract
Purpose: Early detection is essential for treatment plans before
onset of metastatic disease. Our purpose was to demonstratefeasibility to detect and monitor estrogen receptor 1 (ESR1) genemutations at the single circulating tumor cell (CTC) level inmetastatic breast cancer (MBC).
Experimental Design: We used a CTC molecular characterization approach to investigate heterogeneity of 14 hotspot
mutations in ESR1 and their correlation with endocrine resistance. Combining the CellSearch and DEPArray technologies
allowed recovery of 71 single CTCs and 12 WBC from 3
ER-positive MBC patients. Forty CTCs and 12 WBC were
subjected to whole genome ampliﬁcation by MALBAC andSanger sequencing.
Results: Among 3 selected patients, 2 had an ESR1 mutation(Y537). One showed two different ESR1 variants in a single CTC

and another showed loss of heterozygosity. All mutations weredetected in matched cell-free DNA (cfDNA). Furthermore, one
had 2 serial blood samples analyzed and showed changes in both
cfDNA and CTCs with emergence of mutations in ESR1 (Y537S
and T570I), which has not been reported previously.
Conclusions: CTCs are easily accessible biomarkers to monitor
and better personalize management of patients with previously
demonstrated ER-MBC who are progressing on endocrine therapy. We showed that single CTC analysis can yield important
information on clonal heterogeneity and can be a source of
discovery of novel and potential driver mutations. Finally, we
also validate a workﬂow for liquid biopsy that will facilitate
early detection of ESR1 mutations, the emergence of endocrine
resistance and the choice of further target therapy. Clin Cancer

Introduction

chemotherapy (2). According to the clonal theory of tumor
evolution, cancer is an evolving process (3), and the selective
pressure exerted by multiple lines of treatment may lead toselection of much more aggressive subclone populations or eventhose with an acquired drug resistance (4).
About 75% of breast cancers express the estrogen receptor (ER);and, acting on this signaling pathway is a key treatment strategy.
The main endocrine therapeutic approaches are: (i) selective ER
modulators (SERM); (ii) inhibitor of aromatase (AI); and (iii)selective ER downregulators (SERD; ref. 5). However, in 20% to
25% of metastatic breast cancer (MBC) patients, endocrine therapy failure has been reported after several lines of treatment, andnew targeted therapies have been approved to be combined withhormone therapy (6–8). Several molecular mechanisms of resistance may be involved, including downregulation and posttranslational modiﬁcation of the ER encoded by the ESR1 gene (9).
Because ESR1 mutations are rare, occurring in only 1% of primarybreast cancer, their ability to confer endocrine resistance has been
speculated for many years (10). However, in metastatic tissues theincidence of such mutations is estimated at 20% (11). In the last
several years, 14 ESR1 point mutations have been reported,
mainly localized in the ligand-binding domain (LBD) including
3 hotspot mutations in codons 380, 537, and 538. Functional
study of these mutations showed an ER ligand-independent
activity, highlighting their role in acquired endocrine resistance(12, 13). Therefore, genomic characterization of distant metastasis may provide clinically useful information for the selection ofBreast cancer is the most frequent cancer and the leading causeof cancer-related death among women. Despite advances inprevention, diagnosis, and adjuvant treatment, about 30% ofbreast cancer patients develop metastatic disease (1). Recent
advances suggest that the presence of different tumor cell clones
plays an important role in metastatic progression and resistance to1
Department of Cancer Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia Pennsylvania. 2Institute of Biochemistry andClinical Biochemistry, Laboratory of Clinical Molecular and Personalized Diagnostics, Catholic University of the Sacred Heart, Rome, Italy. 3Department ofMedicine-Hematology and Oncology, Robert H. Lurie Comprehensive Cancer
Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois.
4
Department of Molecular Medicine, Sapienza University, Rome, Italy.

Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Current address for G. Rossi: European Institute of Oncology, Division ofThoracic Oncology, Milan, Italy.
Corresponding Authors: Paolo M. Fortina, Sidney Kimmel Cancer Center, Thomas
Jefferson University, 233 S 10th street 1009-BLSB Philadelphia, PA 19107. Phone:215-955-0683; Fax: 215-503-9245; E-mail: paolo.fortina@jefferson.edu; Massimo
Cristofanilli, Robert H. Lurie Medical Research Center, 303 E Superior, Chicago IL
60611. Phone: 312-695-6180; E-mail: massimo.cristofanilli@nm.org
doi: 10.1158/1078-0432.CCR-17-1173
2017 American Association for Cancer Research.

Res; 23(20); 6086–93. 2017 AACR.

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Published OnlineFirst July 5, 2017; DOI: 10.1158/1078-0432.CCR-17-1173

ESR1 Mutations in Single CTC

Translational Relevance
Current treatment strategies, including single-agent endocrine agents or combinations with CDK4/6 inhibitors or
mTOR inhibitors, have increased capabilities of effective treatment of patients with hormone-receptor–positive metastatic
breast cancer. Primary or secondary endocrine resistance is amajor clinical challenge in the management of patients withadvanced hormone-receptor–positive breast cancer because it
is a dynamic phenomenon including development of estrogen-receptor (ESR1) mutations. The evaluation and longitudinal monitoring of endocrine resistance including enumeration of CTCs, measurement of heterogeneous estrogen-receptor expression in those cancer cells and detection of ESR1
mutations allows real-time molecular monitoring allowing
to adapt treatment modalities with potential impact on
outcome.

speciﬁc therapeutic treatments (14, 15). Even though genetic
testing on repeated metastatic biopsies may not be representativeof the whole tumor mass and leads to an underestimation oftumor heterogeneity (16).
Liquid biopsy using either circulating tumor cells (CTC) or cellfree tumor DNA (ctDNA) has become one of the most sensitive
approaches to monitor tumor molecular evolution (17, 18). CTCs
can be isolated noninvasively over time (19) and over the past
decade, the prognostic value of CTCs has been shown in metastatic breast, colorectal, and prostate cancer (20–26). Signiﬁcantadvances in cancer diagnosis and in the evaluation of diseaseprogression and treatment can be reached with single CTC analysis because of the improvements made in single-cell genomics
analysis (27). Currently, there are few techniques available forsingle cell isolation, including micromanipulation, laser microdissection, and high-throughput ﬂuorescence-activated cell sorting (FACS). These approaches have several disadvantages, including inadequate detection sensitivity for CTC population, a
required high number of cells as a starting population and aremanual and laborious methods (28–30).
We decided to study the molecular features of CTCs in patientswith hormone-receptor–positive (HRþ) MBC receiving endocrine
therapy. We planned to validate a laboratory workﬂow for single
CTC detection, isolation and molecular analysis by combining thesequential use of CellSearch (20–26), DEPArray systems (31, 32)
and MALBAC techniques (33). The purpose of this study was toinvestigate the incidence and heterogeneity of ER expression andto evaluate the detection of ESR1 mutations in individual CTCs.
We also planned to compare our single CTC data with matched
cfDNA.

Materials and Methods
Patients and sample collection
Thirty MBC patients were enrolled at the Department of Medical Oncology, Thomas Jefferson University in Philadelphia,
between February and September 2015. Only patients with a
primary ER-positive MBC were included. Clinical parameters
included sex, age at surgery, differentiation grade, lymph nodemetastasis, distant metastasis, TNM stage, and histology. Progesterone receptor (PR) and ER status of the primary tumor and of

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available metastases were recorded. All subjects gave informed
consent, and the study was approved by the Institutional Review
Board. For each patient, 10 mL of blood was collected in two
CellSave tubes (Veridex, LLC) for enrichment, enumeration, andmolecular characterization of CTCs using the FDA-approved
CellSearch System. All samples were taken at least 5 days after
the last treatment. Matching primary tumor tissues were tested forthe presence of mutations in ER receptor before starting hormonal
therapy.
Cell lines
Two human breast cancer cell lines (MCF-7 and FC-IBC-02)
and a prostate cancer cell line (C4-2) were used to validate wholegenome ampliﬁcation experiments. MCF-7 cells were maintainedin DMEM containing 10% (v/v) FBS, 100 units/mL penicillin and100 mg/mL streptomycin. FC-IBC-02 primary cells were isolated
from pleural effusion of IBC patients and cultured in Ham's F12with 10% (v/v) FBS, 5 mL insulin and 100 mg/mL of hydrocortisone with antibiotic antimicotic. The C4-2 cell line was cultured in
RPMI with 2.5% to 10% (v/v) FBS as previously described (34).
All cell lines were maintained in T-25 or T-75 ﬂasks using prescribed cell culture conditions [5% (v/v) CO2, 37 C].
Quality control and experimental procedure validation
Single tumor cells were obtained from MCF-7 and FC-IBC-02
cell lines through micromanipulation and from C4-2 cell line byserial dilution. All collected cells were processed for MALBAC.
Meanwhile, 30 pg of genomic DNA carrying the V600E mutationin the BRAF gene was used as MALBAC-positive control for all
WGA products. To validate the CellSearch ability to capture CTC,
100 C4-2 cells were spiked into a healthy donor blood sample.
Captured C4-2 cells together with WBC from the healthy donor
were loaded on the DepArray cartridge to achieve single cellisolation and capture.
DEPArray system is a semiautomated system that allows theisolation of rare ﬂuorescently labeled cells. An electric ﬁeld isgenerated on the surface of a silicon chip directly interfaced to a
microﬂuidic chamber containing the cell suspension and an arrayof electrodes. Each electrode can be programmed to achieve a cageof dielectrophoresis, inside of which single CTC can be trapped
and then analyzed individually.
Individual C4-2 and WBC cells were subsequently MALBAC
ampliﬁed and screened for 7 known mutations in the AR, CDH1,
PIK3C3, NCOR2, ERBB2, CDK4, and ETV1 genes by Sanger
sequencing. C4-2 and WBC genomic DNA from a healthy donor
also were used to conﬁrm the 7 variants by Sanger sequencing.
Enrichment, immune labeling, and enumeration of CTCs
Standard CellSearch protocol for CTCs enrichment and enumeration was used according to the manufacturer's instructions.
Brieﬂy, CTCs were enriched on the CellTracks Autoprep using
ferroﬂuid conjugated with EpCAM antibody. Cells were stainedwith ﬂuorescently labeled monoclonal antibody for cytokeratin
CK8-, CK18-, CK19-FITC as well as for leukocyte common antigen
CD45-APC and nuclear-stained with DAPI. Moreover, ER expression on MCF-7 cells and CTCs was assessed by staining the cellswith a PE-conjugated anti-ER nuclear antibody.
Because the DEPArray system (Silicon Biosystem) provides theanalysis of only 66% of the loaded volume, to optimize single
CTC recovery rate, only patients exhibiting >20 CTCs (ERþ andER) were processed.

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Paolillo et al.

Single CTC isolation
Brieﬂy, for each CellSearch enriched sample, 13 mL were
loaded with 325 mL manipulation buffer (SB115, Silicon
Biosystem) into an A300K cartridge. Approximately 8.6 mL of
the sample is de facto dielectrophoretically processed in whichcells are individually trapped in cage. The cartridge is thenscanned by an automated ﬂuorescence microscope and cells
detected by DAPI staining. Three different populations of cells
were isolated: (i) ERþ CTCs, deﬁned as ER-positive, CK-8,
CK-18, CK-19–positive, CD45-negative; (ii) ER CTCs, deﬁned
as ER-negative, CK-positive, CD45-negative; and (iii) WBCs,deﬁned as CD45-positive, ER, and CK-negative. Each cell wascollected individually, washed two times in PBS and stored at
80 C or immediately lysed in accordance with MALBAC
protocol (35). To minimize DNA contamination in the same
isolation cage containing the individual cell, an aliquot of theelution buffer from the single cage was MALBAC-ampliﬁed andsubjected to DNA Sanger sequencing. No mutation was
detected on all elution buffer reactions. Moreover, for eachsequencing run, a no template control was tested.

Table 1. Clinicopathologic features of 30 MBCs
Clinicopathologic features
DetailAgeMedianMinimum
Maximum
Histology
DuctalLobular
Other
MissingType
IBC
No IBCER
Positive
Negative
PR
Positive
Negative
HER2
Positive
Negative
Missing
Metastasis sites
n¼1
n>1
a
ER metastatis
Positive
Negative
PR metastasisa
Positive
Negative

Whole genome ampliﬁcation
Cell lysis and genome ampliﬁcation was performed using the
MALBAC kit (Yikon Genomics YK001A/B version 1302.1; ref. 33),
following the manufacturer's instructions. A negative no templatecontrol (NTC), a blank control (SB115), and a MALBAC-positive
control were used for each MALBAC reaction. WGA products werethen puriﬁed according to the Agencourt AMPure XP bead kit(Beckman Coulter) manufacturer's protocol (36) and QC using
Qubit dsDNA High Sensitivity Assay kit (ThermoFisher). WGA
products were run on 0.8% (v/v) agarose gel and checked for
expected distribution in size (300–2,000 bp).

Abbreviation: IBC, inﬂammatory breast cancer.
a
ER and PR status immunohistochemistry on available metastatic lesions.

Sanger sequencing
Sanger sequencing was performed to genotype all WGA products as well as 14 hotspot mutations in the ESR1 gene found in
MBC tissues and related controls on an AB 3730 following the
manufacturer's protocol. Detailed PCR conditions (Tm) andprimer sequences are available in Supplementary Methods(Table 1). Sequences were analyzed and genotyped by SeqScape
v3.0 analysis software (ThermoFisher).

Results
To investigate whether detection of ESR1 mutations in individual CTCs in MBC patients could be used as a tool to enable
monitoring of the metastatic burden for clinical decision-making,a 4-step protocol was implemented with the following workﬂow:(i) CTC enrichment, (ii) single cell isolation, (iii) whole genome
ampliﬁcation, and (iv) Sanger sequencing (Fig. 1).
Patient and pathological features
A cohort of 30 MBC patients was characterized by a median ageof 56 years. Patients (36.6%) showed evidence of one single
metastatic lesion, while 64% showed more than one at the timeof the ﬁrst draw. Clinical and pathological features of primary andmetastatic tumor tissues are summarized in Table 1. Among thetotal number of patients, 28 had histologically conﬁrmed ER
positivity even at metastatic sites. At the time of surgery, primarytumor tissues were investigated for presence of ER mutations andall harbored a wild-type genotype.

6088 Clin Cancer Res; 23(20) October 15, 2017

n
56
34
79
18
7
2
3
16
14
30
0
20
10
2
24
4
11
19
28
2
20
10

Enrichment, isolation, and genome ampliﬁcation ofindividual CTCs
Enrichment and enumeration of CTCs performed on CellSearch involved a total of 50 blood samples taken from the 30
patients enrolled. The number of CTCs based on ER expression(Fig. 2) for each patient (ID) are shown in Table 2. Overall, theaverage of total CTCs enumerated was 80, with a maximum of
1,375 cells. Samples (22%) were negative (no CTCs) for thepresence of CTCs, deﬁning a group of patients currently responsive to current treatment. The remaining 39 samples were divided
into two groups depending on the established cutoff of 5 CTCs
(37) used to identify patients with high risk of disease progression(12 samples <5 CTCs vs. 27 samples 5 CTCs). Only 4 of the 50
samples analyzed showed a number of CTCs greater than or equal
to 20 and were processed on the DEPArray. Seventy-one single
CTCs and 12 white blood cells (WBC) were retrieved. Forty ofthese CTCs and all the WBCs were subjected to WGA. The numberof CTCs (subdivided in ERþ or ER) isolated for each individual
patient and the corresponding number of selected cells for WGA
are summarized in Table 3. Samples processed on DEPArray
showed between 21% and 30% of CTCs recovered.
Preclinical validation of single-cell genome
ampliﬁcation and analysis
Validation of single-cell genome ampliﬁcation was conducted on 30 individual single cells. DNA-positive controls,
after MALBAC ampliﬁcation showed heterozygosity for the
BRAF V600E (c.1860T>a) mutation (data not shown), as
expected. No other BRAF mutations were found in all thewild-type single cells analyzed, demonstrating feasibility of the
protocol.
Following spiking, enrichment and immune labeling, 91 positive CTCs were detected by CellSearch. Sixty-ﬁve percent of these
CTCs were identiﬁed on the DEPArray and ﬁnally 10 individual
cells were recovered and subjected to whole genome ampliﬁcation. In addition, 10 WBCs were recovered and subjected to WGA,
as negative control. The sequences obtained from all the C4-2

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ESR1 Mutations in Single CTC

Figure 1.
Study workﬂow. The total number of
CTCs analyzed from 3 different
patients who showed a number ofERþ CTCs > 20. For each patient, 3
WBC were recovered as negative
controls. One patient was tested twice
because of disease progression.

cells (single or pooled) revealed all 7 carried known mutations (AR/T878A, CDH1/P94T, CDK4/P110L, ErbB2/E930D,
ETV1/G207E, NCOR2/L167P, PIK3C3/F524C; data not shown).
Sequences obtained from the 10 WBCs from healthy donor's
buffy coat showed wild-type genotypes for all variants tested.
ESR1 mutational analysis in single CTCs
ESR1 mutation analysis was successfully performed on all
single cells isolated. All mutations were located within theligand-binding domain of the ESR1 gene in exons 4, 5, 6, 7, and
8 (Fig. 3). All 12 WBCs showed a wild-type genotype conﬁrming
the absence of mutations in the germline. Overall, we found ESR1
mutations in a total of 8 CTCs belonging to 2 MBC patients. Highlevels of intra- and intertumor genetic heterogeneity in ER-positive CTCs populations was also revealed. The remaining 32 CTCs
analyzed showed a wild-type ESR1 genotype. For patient ID20, a
total of 5 single CTCs were analyzed and all showed wild-type
phenotype. Patient ID19 exhibited a heterogeneous ESR1 genotype in their CTC populations. Among the ERþ CTC population,
we detected 3 different genotypes: (i) one single wild-type CTC;(ii) 3 CTCs heterozygous for a single mutation (Y537S) in exon 8(Fig. 3a); (iii) one single CTC homozygous for the same Y537S(LOH; Fig. 3b). Matching cfDNA was tested, conﬁrming the Y537S
mutation at 0.25% allele frequency. Patient ID10 was the only one
who had 2 serial blood samples taken, 3 months apart. The ﬁrst
sample showed an ESR1 wild-type genotype in all the 12 CTCs
recovered. The second showed a wild-type genotype in all 8 ER
CTCs, and in 4 of the ERþ CTCs recovered. Three CTCs were
heterozygous for the Y537S mutation, whereas the remaining ERþ

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CTC harbored 2 different mutations in exon 8. Other than the
Y537S we found a new mutation, not reported until now, theT570I (Fig. 3c). Also, in this case, data were compared with thoseobtained on matching cfDNA. No mutation was detected at theﬁrst draw, while Y537S was detected at the second sampling at apercentage of 0.18%.
Correlation between ESR1 mutation and patients' treatment
Patient ID19 with the Y537S mutation had only one samplingfor CTC enumeration and circulating free tumor DNA (cfDNA)
analysis. The ﬁrst diagnosis of inﬂammatory ductal breast cancer
was made in 2010, and the patient opted for holistic remedies.
In 2011, the patient developed ascites and pleural effusions
and started a chemotherapeutic treatment (docetaxel–cytoxan)for 6 months, followed by endocrine therapy with aromasin.
In 2014, the patient was subjected to 6 cycles of Doxil and
Faslodex, but in January 2015, a liver metastasis was found. This
progression to metastatic disease during treatment indicated a
failure in the therapeutic approach. The analysis of CTCs andcfDNA conﬁrmed this suspicion, with the ﬁnding of the Y537Sactivating mutation.
Patient ID10 reported two mutations, the Y537S and the newly
reported T570I. This case report shows how the monitoring of
ESR1 mutations can be crucial to monitor and predict disease
evolution. First diagnosis was made in 2011, which was followed
by a mastectomy. In 2012, she was irradiated and subsequently
treated with tamoxifen. Due to poor tolerability of the drug and tothe occurrence of bone metastasis, the patient switched endocrine
therapy, examestane followed by fulvestrant. At the time of theClin Cancer Res; 23(20) October 15, 2017

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Paolillo et al.

Figure 2.
ER nuclear expression. Representative
CellSearch images for (A) ER
expression in the MCF-7 cell line
(events 1, 2, 3 are ER, events 4 and 5
are ERþ); (B) CTCs from patient
sample (all events are ERþ).

ﬁrst draw, she was negative for ESR1 mutations, but a highincrease in the number of CTCs was found compared withbaseline CTCs count (80 vs. 54). The second sampling was taken
only 1 month apart and was positive for the Y537S-activating
mutation. Furthermore, a third CTC count was made after afurther therapy switch to combination with palbociclib, showing
a relative decrease in total number of CTCs (43 vs. 80), but analmost unchanged number of the ERþ population (23 vs. 28). The
prospective clinical evidence clearly indicate that the patient did
not beneﬁt from fulvestrant at time of ESR1 mutation detection.
Instead, she beneﬁted from the prompt switch to a combined
therapy of palbociclib and fulvestrant, a highly effective regimen
evaluated in the prospective, randomized, phase III study
PALOMA-3, whose beneﬁt appears irrespective of common genomic abnormalities such as ESR1 and PI3KCA (38, 39).

Discussion
The frequency of ESR1 mutations in breast cancer is a matter ofintense debate for the potential clinical utility of this information.

6090 Clin Cancer Res; 23(20) October 15, 2017

Primary ESR1 mutations are relatively rare in primary tissue, up to
7% of specimens analyzed with very low allele frequencies(0.07%–0.2%), when compared with a much higher detection
in patients with metastatic disease (11). The ﬁrst study on thedetection of ESR1 mutations in patients that were exposed to
endocrine therapy was conducted on metastatic biopsies andmatched cfDNA samples (37). Two hotspot mutations in codons
537 and 538 of the ESR1 gene were investigated by digital PCR(dPCR). Those ﬁndings showed monitoring ESR1 mutations by
dPCR was feasible, but not all mutations found in the metastatic
biopsies were detected also in matched cfDNA (40). In our study,
we used the analysis on cfDNA as a validation of the results foundat the CTC level. All mutations found in cfDNA were conﬁrmed in
the ERþ CTC population. In CTCs, we detected a new mutation incodon 8 (T570I) that was not detected in cfDNA. On the other
hand, ER expression in CTCs showed a wide heterogeneous status.
Most samples positive for CTCs showed a mixed population (ERþand ER), but 5 samples positive for CTCs were negative for ER
expression. These results suggest that analysis of both CTCs andcfDNA can be a useful guide in clinical practice.

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ESR1 Mutations in Single CTC

Table 2. CTC number assessed by CellSearch for each patient, based on ER surface expression
Patient ID
CTCþ/ERCTCþ/ERþ
Total
Patient ID
1
0
0
0
15a
2a
1
3
4
3
1
4
16a
3
0
0
0
4
0
0
0
17
4
1
0
1
18a
5a
2
2
4
0
2
2
19
6
0
6
20a
6
3
0
3
7a
3
0
3
21
2
2
4
22a
8
0
5
5
9
2
2
4
23
10a
11
43
54
24
22
58
80
25
10
13
31
44
26
11
0
0
0
27
12
5
0
5
28
13
1
0
1
29
14
2
3
5
30

CTCþ/ER
9
0
147
410
0
13
5
19
12
13
0
1,361
931
0
0
22
19
0
0
0
0

CTCþ/ERþ
18
1
0
0
2
15
14
33
44
9
0
14
0
0
0
18
9
0
0
0
0

Total
27
1
147
410
2
28
19
52
56
22
0
1,375
931
0
0
40
28
0
0
0
0

a

More than one draw was performed during the enrollment period.

Our study is the ﬁrst in which the combined systems (CellSearch and DEPArray) were applied to assess both ER expression and all 14 ESR1 hotspot mutations by the MALBAC singlecell ampliﬁcation method. The combined approach, CellSearchand DEPArray, was tested already on cancer patients' samples(41–43). In a previous study, 510 CTCs were isolated from 66
MBCs. Thirty-seven CTCs were subjected to adaptor–ligationmediated whole-genome ampliﬁcation and subsequently analyzed for the expression of the ErbB2 gene and for analysis of
two hotspots in PIK3CA (exons 20 and 9; ref. 41). They
demonstrated applicability of that workﬂow and also foundsome heterogeneity between the analyzed CTCs and primarytumor. Another group studied the entire population of CTCs
and white blood cells enriched from the CellSearch system togenotype ESR1, PIK3CA, TP53, FGFR1, and FGFR2 genes, by anext-generation sequencing (NGS) panel. Analysis of suchmarkers was also done on cfDNA and 4 repeated samples overtime during patient therapy monitoring. Two patients showedchanges at the level of ESR1 gene mutations detected in cfDNA,
only one in cfDNA and CTCs. This discordance can be
explained by the fact that CTCs were analyzed as a pool with
WBCs, where the predominant component is wild-type (42).
Only recently the same group published a new study where the
NGS panel was also performed on single CTC isolated by
DEPArray. The purpose of the study was to determine whether
cfDNA can be compared with single CTC analysis to detecttumor mutation heterogeneity (44).
Our work is the ﬁrst to evaluate detection of all activating ESR1
mutations among the LBD, at the single circulating tumor celllevel in MBC patients. We also monitored the acquisition of

endocrine resistance and validated data conﬁrming mutations inmatched cfDNA. Of the 4 samples processed and corresponding
to 3 different patients, 2 had at least one mutation that was also
conﬁrmed in cfDNA, but not in primary tissue. In both samples,the mutation detected was Y537S, positioned in exon 8 of the LBD
domain of ER, as well as one of the most common mutations
found in metastatic lesions. This mutation was present only insome of the CTCs from the same patient, highlighting the importance of single cell analysis instead of the previously poolingstrategy (40, 42).
The role of wild-type tyrosine 537 and the effects of a number ofpossible amino acid substitutions have been thoroughly investigated (13). This site is located in domain E, ligand binding, andrecognition region, containing the functional transcription activating domain ligand dipendene-2 and involved in the regulationof ER transcriptional activity. Among all the substitutions tested,
the Y537S was the only one that showed 100% activity of thereceptor in the absence of ligand (45). A recent study showed thatsuch activity can be partially reduced by increasing the tamoxifen
or fulvestrant doses, a possible strategy to avoid endocrine resistance (46). This mutation could deﬁnitely be one of the maincauses of poor or inadequate response to hormone therapy.
Finally, we also demonstrate how our workﬂow allows investigation of intratumor heterogeneity. A loss of heterozygosity(LOH) was detected in one single CTC. We cannot totally exclude
that this may be due to a technical error deriving from WGA.
Analyzing a higher number of CTCs for each patient has in partsolved this issue. For the ﬁrst time, in our work, MALBAC has beenused in combination with the CellSearch and the DEPArraysystems. None among the various techniques can ensure theTable 3. Summary of the number of CTCs detected, isolated, and selected to perform WGA
No. of CTCs on
No. of CTCs on% of CTCs
Patient ID
CellSearch ER(þ, )
DEPArray ER(þ, )
recovered19
56 (44, 12)
17 (8, 9)30
2052 (33, 19)
11 (6, 5)
21
54 (43, 11)
15 (11, 4)
28
10a80 (28, 52)
19 (8, 11)2310aNo. of WGA
performed on ER(þ, )
7 (5, 2)5 (3, 2)12 (8, 4)
16 (8, 8)

No. of WGA
performed on WBCs
3
3
3
3

a

Two draws were performed during the enrollment period; a third count on CellSearch was performed and showed a decrease in the total number of CTCs (44), but analmost stable number of ERþ CTCs (23).

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Published OnlineFirst July 5, 2017; DOI: 10.1158/1078-0432.CCR-17-1173

Paolillo et al.

Figure 3.
DEPArray imaging reports and Sanger sequence result. A, Example of Y537S heterozygous mutation. B, The only homozygous Y537S mutation found in patient ID10.
C, T570I novel, unreported mutation found in patient ID10.

absence of errors like false positive/negative results or allelicdropout (ADO). Several comparison studies have shown thateach WGA technique has its own beneﬁts and drawbacks. The
main advantages of MALBAC are associated with reduced ADO
and PCR bias, high ampliﬁcation efﬁciency even of GC-richregions and high genome coverage (up to 90%; ref. 47). Moreover,
an improved ability of MALBAC in SNP variant identiﬁcation has
been recently reported, with better performance in uniformity andreproducibility (48). Other methods, like SurePlex, allow for abetter copy-number alterations (CNA) detection, with a more
uniformity of ampliﬁcation across the genome (49). These observations led us to endorse MALBAC as the technique to be used ingenotyping the ESR1 gene. MALBAC is therefore conﬁrmed by us
to be a reliable WGA method to address single CTC molecular
proﬁling.
In conclusion, this study demonstrated the feasibility of our
protocol to detect and monitor ESR1 gene mutations at the single
CTC level in MBCs. Early detection is essential to set the correct
treatment plan for patients, before onset of metastatic disease. In
addition, analysis of individual CTCs could allow identiﬁcation of
new potentially driving mutations or even new genes involved inresistance. Further studies with larger numbers of patients are
required to make this approach of use in the clinic.

Authors' Contributions
Conception and design: C. Paolillo, K. Knudsen, M. Cristofanilli, P. Fortina
Development of methodology: C. Paolillo, Z. Mu, M. Cristofanilli
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): C. Paolillo, M.J. Schiewer, T. Nguyen, L. Austin
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): C. Paolillo, T. Nguyen, M. Cristofanilli
Writing, review, and/or revision of the manuscript: C. Paolillo, Z. Mu,
M.J. Schiewer, E. Capoluongo, M. Cristofanilli, P. Fortina
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): C. Paolillo, Z. Mu, E. Capoluongo
Study supervision: M. Cristofanilli, P. Fortina
Other (collection and organization of the data in a database): G. Rossi

Acknowledgments
We wish to thank Dr. Saul Surrey for helpful and valuable suggestions.

Grant Support
This work was supported in part by NIH–National Cancer Institute Core
Grant P30CA56036 (P. Fortina), Thomas Jefferson University Institutional
funds (P. Fortina), by the Catholic University of the Sacred Heart Institutional
funds (C. Paolillo).
The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disclosure of Potential Conﬂicts of Interest
M. Cristofanilli is a consultant/advisory board member for Vortex. No
potential conﬂicts of interest were disclosed by the other authors.

Received April 26, 2017; revised June 20, 2017; accepted June 30, 2017;
published OnlineFirst July 5, 2017.

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Published OnlineFirst July 5, 2017; DOI: 10.1158/1078-0432.CCR-17-1173

Detection of Activating Estrogen Receptor Gene (ESR1) Mutationsin Single Circulating Tumor Cells
Carmela Paolillo, Zhaomei Mu, Giovanna Rossi, et al.
Clin Cancer Res 2017;23:6086-6093. Published OnlineFirst July 5, 2017.

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