Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non-small cell lung cancer

Abstract

Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3-15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0-2 cells/1.88 mL of blood). The latter range was validated as the 'false positive' cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing.

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www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 17
Concordance of anaplastic lymphoma kinase (ALK) gene
rearrangements between circulating tumor cells and tumor in
non-small cell lung cancer
Chye Ling Tan1,*, Tse Hui Lim1,*, Tony KH Lim1, Daniel Shao-Weng Tan2, Yong Wei
Chua1, Mei Kim Ang2, Brendan Pang3, Chwee Teck Lim4,5, Angela Takano1, Alvin
Soon-Tiong Lim1, Man Chun Leong6 and Wan-Teck Lim2,7
1 Department of Pathology, Singapore General Hospital, Singapore
2 Department of Medical Oncology, National Cancer Center Singapore, Singapore
3 Department of Molecular Oncology, National University Health System Singapore, Singapore
4 Faculty of Engineering, Department of Biomedical Engineering, National University of Singapore, Singapore
5 Mechanobiology Institute, National University of Singapore, Singapore
6 Clearbridge Biomedics Pte Ltd, Singapore
7 Institute of Molecular and Cell Biology, Singapore
* These authors have contributed equally to this work
Correspondence to: Wan-Teck Lim, email: dmolwt@nccs.com.sg
Keywords: ALK-gene rearrangement, circulating tumor cells, uorescent in-situ hybridization, lung cancer, molecular diagnosis
Received: November 14, 2015 Accepted: February 28, 2016 Published: March 16, 2016
ABSTRACT
Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung
cancer (NSCLC) is routinely evaluated by uorescent in-situ hybridization (FISH)
testing on biopsy tissues. Testing can be challenging however, when suitable tissue
samples are unavailable. We examined the relevance of circulating tumor cells (CTC)
as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC
samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung
cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts,
and to compare ALK rearrangement patterns. Blood samples were enriched for
CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-
positive NSCLC patients (3–15 cells/1.88 mL of blood) compared with ALK-negative
NSCLC patients and healthy donors (0–2 cells/1.88 mL of blood). The latter range
was validated as the ‘false positive’ cutoff for ALK FISH testing of CTCs. ALK FISH
signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases.
Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue
treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL
blood at baseline, partial response and post-progression time points, respectively.
Furthermore, ALK FISH rearrangement suggestive of gene copy number increase
was observed in CTCs following progression. Recapitulation of ALK rearrangement
patterns in the tumor on CTCs, suggested that CTCs might be used to complement
tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue
biopsy samples are unavailable for testing.
INTRODUCTION
Lung cancer accounts for about 13% of all cancer
diagnoses and remains the leading cause of death by
cancer in the world [1], with almost 70% of patients
diagnosed with locally advanced or metastatic disease at
presentation [1, 2]. Non-small cell lung cancer (NSCLC)
accounts for approximately 85% of all cases of lung
cancer and is associated with poor prognosis [2]. The
5-year overall survival rate for NSCLC across all stages
is only 21% and is even lower (~5%) for stages IIIB and
IV [1, 3].

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Oncogenic ‘driver mutations’ have now been
identied in various subsets of NSCLC [4-6]. Of these
drivers, somatic mutations in epidermal growth factor
receptor (EGFR) and anaplastic lymphoma kinase
(ALK) [5-7] are the most frequently described. In 2007,
researchers identied the presence of a chimeric ALK
protein with broblast-transforming properties that was
formed following fusion of the echinoderm microtubule-
associated protein-like 4 (EML4) and ALK genes [6].
EML4-ALK subverts intracellular signaling pathways to
promote tumor cell survival and growth [8]. The overall
incidence of ALK gene rearrangement in NSCLC ranges
between 0.4% and 13.4%, and is similar in both Asian and
Western populations [9]. This discovery resulted in the
accelerated development and approval by the U.S. Food
and Drug Administration (FDA) of the ALK-targeting
tyrosine kinase inhibitors (TKIs) crizotinib (Xalkori®,
Pzer, New York, USA) in 2011, and ceritinib (Zykadia™,
Novartis, Basel, Switzerland) in 2014 to treat patients with
metastatic NSCLC who express the abnormal ALK gene
[10, 11].
The true therapeutic benet of novel molecules
targeting the mutant ALK fusion protein in NSCLC relies
on identifying the right patient population for treatment,
and on detecting the emergence of tumor resistance. The
American Society of Clinical Oncology (ASCO) endorsed
the joint College of American Pathologists (CAP)/
International Association for the Study of Lung Cancer
(IASLC)/Association for Molecular Pathology (AMP)
clinical practice guideline on EGFR and ALK molecular
testing for patients with lung cancer, which holds that an
ALK uorescent in-situ hybridization (FISH) assay using
dual-labeled break-apart probes is the preferred testing
methodology to detect ALK gene rearrangement [12].
The accuracy of testing nonetheless depends on the
quality of tumor biopsies. Approximately 50% of NSCLC
patients who undergo re-biopsy for determination of
resistance after rst-line chemotherapy have insufcient/
non-diagnostic biopsy specimens or cytology samples
available for molecular testing [13]. Biopsy is invasive and
repeating the procedure is not always feasible due to safety
concerns and general unwillingness of patients, among
other reasons [14]. Furthermore, lung adenocarcinomas
are heterogeneous with a diverse and ever-evolving
genetic and epigenetic makeup that contributes towards
treatment resistance [15]. These barriers to biopsy
collectively pose a challenge to track oncogene activity in
real-time over the course of treatment. There is a need for
a minimally invasive assay for tumor molecular proling
and continuous treatment monitoring in order to provide
timely and tailored cancer treatment.
Circulating tumor cells (CTCs) released from
the primary tumor site into the circulation represent a
potential means of non-invasively isolating tumor cells for
ALK FISH testing and other molecular characterizations.
Recent data supports the role of these renegade cells as
seeds of cancer metastases [16, 17]. They may recapitulate
the phenotypic heterogeneity and molecular signatures of
the primary tumor, as well as that of metastatic lesions
[18-20]. While their presence and prevalence in blood are
often associated with poor prognosis [21], CTCs may hold
further relevance as an alternative tumor source, which
can complement existing tissue-based diagnostic tests,
especially when biopsy material is absent or inadequate. In
patients with lung adenocarcinoma, hypothesis-generating
studies have strongly suggested that ALK status could be
determined based on testing of CTCs, with comparable
results as testing of tumor tissues [22, 23].
The key challenge of CTC-based testing is
the enrichment and isolation of these cells within an
acceptable timeframe. Various technical approaches
have been used to isolate these CTCs [19, 24, 25].
They can be broadly categorized into antibody afnity-
based, imaging-based and size-based techniques [26].
The only current US FDA-approved CTC capturing
technology utilizes EpCAM immunomagnetic means to
isolate EpCAM-positive CTCs for prognostic purposes
and would inadvertently miss out on EpCAM-negative
CTCs [27, 28]. As a predictive biomarker for treatment
monitoring and molecular analysis, it is pertinent to ensure
reliable and reproducible isolation of CTCs of different
phenotypic and molecular subtypes for various pre-and
post-treated patient cohorts [25]. An increasing body
of evidence suggests that non-immunomagnetic-based
CTC technologies can reliably retrieve a comprehensive
population of CTCs for molecular subtyping [19, 28].
Here, we evaluated the feasibility of an antibody-
independent CTC isolation system using lung
adenocarcinomas that have been tested ALK positive as a
model to examine the concordance patterns between CTCs
and tumor tissue, and to determine whether CTCs were
reproducibly detectable in circulation. We further explored
the potential use of CTCs in lung cancer, as a surrogate
for molecular testing of the primary tumor for ALK gene
rearrangement.
RESULTS
Study group
We prospectively recruited 27, mostly late-stage
NSCLC patients, 14 of whom had ALK-rearranged and
12 had wild-type ALK, determined from the initial biopsy
diagnoses. One patient in the cohort, who was not from
Singapore, had an unknown ALK status due to incomplete
referral records. Sixty percent of patients were males. All
ALK-positive patients were non-smokers. Five healthy
donors (three males and two females), aged between 18-
55 years old with no history of cancer, were also recruited
into the control cohort. As ALK translocation in NSCLC

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patients is strongly correlated with a non-smoker or
light smoker status [29], these healthy donors were non-
smokers. The clinicopathologic features of the study group
are summarized in Table 1.
Histopathological analysis of tumor tissues
In the ALK-positive group, three out of 14 tumor
tissues exhibited morphology that is associated with ALK
rearrangements [30]. Histological preparations showed
solid adenocarcinoma with signet ring cells (Figure
1A and 1B), and cribrifom adenocarcinoma with focal
squamoid cells (not shown). Immunohistochemical (IHC)
studies showed strong and diffuse nuclear reaction for
Thyroid Transcription Factor-1 (TTF-1). This nding
conrmed the diagnosis of adenocarcinoma of lung origin
in this particular setting (Figure 1C). Some tumors also
showed focal reaction to periodic acid-Schiff with diastase
(PAS-D) within mucin vacuoles, which is a general
feature of adenocarcinomas, as opposed to squamous cell
carcinomas (Figure 1D) [30].
Concordance in ALK rearrangement pattern
between CTCs and tumor
Following FISH testing on all tumor samples in the
cohort, it was found that ALK-positive tumors harbored
ALK rearrangements with various patterns of abnormality
(Table 2). The majority of the tumor samples harbored
the one fusion (F) and one split orange (R) and green (G)
signal (Figure 2A). The tumor from Patient P5 presented
various ALK rearrangement patterns such as 1F1R1G,
2F1R, 2F2R, 1F1R and 1F2R.
FISH testing was subsequently performed on CTCs
that were enriched and isolated from the matched blood
samples. Data showed that ALK rearrangement patterns
(majority 1F1R1G) observed in primary tumor tissues
were recapitulated on most of the ALK-positive CTCs,
giving an overall concordance rate of over 90% based on
the 1F1R1G fusion pattern (Table 2). In Patient P5 (Table
2), the CTCs were able to recapitulate three out of ve
ALK rearrangement patterns observed in the tumor tissue.
We further observed an overexpression of vimentin
in the tumor samples, along with the control bronchiolar
epithelium (Figure 2B). However, loss of E-cadherin was
not obvious in these samples.
The number of ALK-positive rearranged CTCs
Table 1: Clinicopathological characteristics of patients enrolled in this study
Patient characteristics Cases (%)
N = 27
Age, years 32–76
Sex
Male 16 (59.3%)
Female 11 (40.7%)
Smoking history
Non-smoker 16 (59.3%)
Smoker 5 (18.5%)
Ex-smoker 5 (18.5%)
No info 1 (3.7%)
Clinical staging
IB 1 (3.7%)
IIIA 1 (3.7%)
IIIB 2 (7.4%)
IV 23 (85.2%)
Histological subtype
ALK-positive 14 (51.9%)
Adenocarcinoma (NSCLC) 11 (40.7%)
Unknown subtype (NSCLC) 3 (11.1%)
ALK-negative 12 (44.4%)
Adenocarcinoma (NSCLC) 5 (18.5%)
Unknown subtype (NSCLC) 7 (25.9%)
ALK status unknown 1 (3.7%)
Adenocarcinoma (NSCLC) 1 (3.7%)
Abbreviations: ALK, anaplastic lymphoma kinase; NSCLC, non-small cell lung cancer

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Table 2: Concordance of ALK rearrangement patterns between CTC and tumor in patients with ALK-positive NSCLC
Case number of
patients with ALK-
positive NSCLC
ALK rearranged/ total
cells scored (Tumor)
ALK rearrangement patterns
(% of tumor cells observed with respective patterns)
Tumor CTC
P1 61/100 1F1R1G (100%) 1F1R1G (100%)
P2 45/100 1F1R1G (100%) 1F1R1G (100%)
P3 79/100 1F1R1G (100%) 1F1R1G (100%)
P4 30/100 1F1R1G (100%) 1F1R1G (100%)
P5 61/100
1F1R1G (4.9%)
2F1R (34.4%)
2F2R (3.3%)
1F1R (49.2%)
1F2R (8.2%)
1F1R1G (50%)
2F1R (37.5%)
2F2R (12.5%)
P6 72/100 1F1R1G (29.2%)
1F1R (70.8%) 1F1R1G (100%)
P7 81/100 1F1R (100%) 1F1R (75%)
1F1R1G (25%)
P8 55/100 1F1R1G (100%) 1F1R1G (100%)
P9 77/100 1F1R (31.2%)
1F1R1G (68.8%) 1F1R (100%)
P10 100/100 1F1R (100%) 1F1R (50%)
1F1R1G (50%)
P11 62/100 1F1R1G (100%) 1F1R1G (100%)
P12 77/100 1F1R1G (100%) 1F1R1G (100%)
P13 45/100 1F1R1G (100%) 1F1R1G (100%)
P14 Not available Not available 1F1R1G (100%)
Abbreviations: ALK, anaplastic lymphoma kinase; CTC, circulating tumor cells; NSCLC, non-small cell lung cancer.
Figure 1: Representative appearance of NSCLC adenocarcinoma with signet ring cells features. A. H&E stain showing
solid nests of tumor cells B. Solid with signet ring cells (arrow ) C. Thyroid transcription factor-1 (TTF-1) IHC stain showing strong nuclear
reaction in the signet ring cells. D. Solid tumor showing focal positive reaction for (PAS-D) within mucin vacuoles (arrow).

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Figure 2: High concordance of ALK FISH rearrangements patterns between CTCs and tumors in NSCLC
adenocarcinoma patients. A. Representative ALK FISH rearrangement patterns in CTCs and tumors showing 1F1R1G rearrangement
patterns. Yellow, red and green arrows represent fusion (F), orange (R) and green (G) uorescent signals. B. Representative vimentin (upper
panel) and E-cadherin (lower panel) IHC in tumor and control bronchiolar epithelium (black arrow).

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retrieved from ALK-positive patients was signicantly
enriched compared with ALK-negative patients (p <
0.0001) and healthy donors (p = 0.0003) (Figure 3).
Establishment and validation of ALK break-apart
probes cutoff in ALK-negative samples
ALK testing by FISH in NSCLC tumor tissues
without ALK rearrangement may detect rearrangement-
positive patterns (i.e. split patterns or isolated 3’
patterns) in a fraction of cells [31-33], likely because of
truncation artefact caused by tissue sectioning, or perhaps
a stochastic genomic alteration that does not indicate a
specic gene fusion. ALK FISH testing in formalin-xed,
parafn-embedded (FFPE) NSCLC tumor tissues has a
‘false positive’ cutoff value of 15% to allow for the best
separation between ALK-rearranged and ALK wild-type
cells [31, 33]. However, it is not possible to apply this
guideline in the ALK FISH testing on CTCs because the
number of CTCs in any given blood sample would be too
low.
In our study, we established and validated the ‘false
positive’ cutoff for ALK FISH in CTCs using 12 blood
samples from NSCLC ALK-negative patients and ve
blood samples from healthy donors (Supplementary Data).
Results from the ALK-negative NSCLC cohort scored a
median of two or less positive cells (range 0-2 cells/1.88
mL blood). The result concurred with the numbers
observed for healthy blood samples. In fact, no statistical
difference in ALK-positive cell counts was observed
between ALK-negative NSCLC cohort and healthy donors
(p = 0.0973) (Figure 3). This data established the ‘false-
positive’ cutoff for ALK break-apart probes in CTCs at ≤
two cells per 1.88 mL blood.
Potential clinical applications
Sequential CTC enumeration and FISH was
performed on blood samples from a patient with no
accessible tissue for ALK FISH testing. The index case
was a never smoker male diagnosed with NSCLC. A
transthoracic needle aspiration biopsy was performed on
the right hilar mass to obtain a specimen for histological
analysis. The hematoxylin and eosin (H&E) stain
showed one small cluster of NSCLC cells with strong
nuclear reaction for TTF-1 favoring adenocarcinoma.
Unfortunately, his diagnostic tissue was exhausted and no
further molecular proling could be performed. He did not
Figure 3: Number of cells with ALK rearrangements in ALK-positive NSCLC patients is signicantly higher compared
to ALK-negative and healthy donors. Graph represents statistical analyses of the data on Table S1 using the non-parametric two-
tailed t-test. NS represents not signicant while p value <0.05 were considered signicant.

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respond to EGFR tyrosine kinase inhibitor (TKI) therapy
and had a short duration of response to pemetrexed and
cisplatin. Re-biopsy of the lung and liver tumors was
considered but the patient declined due to the risk of
bleeding. He consented to blood sampling instead; the
sample was subsequently processed as described in the
Methods section.
At baseline, six CTCs displaying a 1F1R1G
pattern were isolated and met the necessary cutoffs for
ALK-positivity (Figure 4A). A trial of crizotinib was
commenced. Conrmatory scans done 3 months after
completion of treatment demonstrated good partial
response in the liver and minor response in the primary
lung tumor, based on RECIST criteria (Figure 4A).
Sequential CTC counts dropped to three cells displaying
the similar ALK rearranged pattern as the baseline. He
continued on crizotinib but unfortunately, his disease
progressed in the liver and the brain 5 months after
treatment initiation (Figure 4A). A post-progression blood
sample showed additional ALK rearrangement patterns
present in his CTCs, which differed from the baseline
patterns. New ALK rearrangement patterns such as 2R2G
and 1F1R appeared, in addition to 1F1R1G, which was
previously present (Figure 4B). The number of ALK-
positive CTCs also increased from three to eleven CTCs
per 1.88 mL of blood post-progression.
DISCUSSION
We successfully captured CTCs using an antibody-
independent CTC isolation system. CTCs were enriched
from the blood samples collected from 27 NSCLC
patients, 14 of whom were ALK-positive. Three of
the cases exhibited solid with signet ring cells pattern
associated with ALK positivity [34-37]. Overall, CTCs
isolated from the ALK-positive patient cohort were above
detectable levels, even among previously treated patients.
The presence of ALK rearrangement in CTCs was
previously analyzed and reported by French groups using
the Isolation by Size of Epithelial Tumor (ISET) system,
Figure 4: An index case suggests that ALK-rearranged CTCs could have clinical application as a diagnostic biomarker
to monitor crizotinib treatment and response. A. CT scan taken at baseline, partial response and progression time points showing
presence of metastatic tumor in liver (arrow). CTC counts and ALK rearrangement patterns for each time point is indicated in the lower
panel. B. Representative images showing 1F1R1G, 2R2G and 1F1R ALK rearrangement patterns following progression on crizotinib
treatment. Yellow, red and green arrows represent fusion (F), orange (R) and green (G) uorescent signals.

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which is also an antibody-free system (Rarecells, Paris,
France) [22, 23]. They characterized 18 ALK-positive
lung tumor and CTC samples showing high concordance
in ALK rearrangement among European patient cohort.
In agreement with our data reported in this study, ‘false-
positive’ signals were similarly observed on CTCs
from ALK-negative samples and a 4 cell/mL cutoff was
eventually established [22, 38].
In a similarly designed study, Pailler et al. [22]
described high concordance in ALK rearrangement
patterns between the CTC and tumor biopsies in 18 ALK-
positive and 14 ALK-negative patients with metastatic
NSCLC. This percentage of concordance is in agreement
with our own results. They reported that all ALK-positive
NSCLC patients in their cohort had 4 or more ALK-
rearranged CTCs per mL of blood. The study did not
include healthy donors to establish a ‘false positive’ cutoff
for ALK FISH testing of CTCs. They further reported that
CTCs harboring the 1F1R1G ALK rearrangement patterns
is associated with epithelial-mesenchymal transition
(EMT) phenotype [22].
Another study had reported that the EMT phenotype
(represented by loss of E-cadherin and expression of
vimentin) was more common in ALK-rearranged tumors
than other genotypes (38.9%, 19.1%, 26.9% and 14.6% of
ALK-rearranged, EGFR-mutated, K-ras mutated and triple
negative tumors, respectively; p = 0.015) [39]. Separately,
expression of vimentin alone was detected in 49.30%
of ALK-rearranged tumors while loss of E-cadherin was
detected in 71.30% [39]. In our study, we also observed
an overexpression of vimentin in the tumor samples in
comparison with the control bronchiolar epithelial tissue.
However, the loss of E-cadherin was not obvious in our
tumor samples, which suggested that some of the tumor
cells retained their epithelial characteristics within a
heterogeneous population of cells. The predominance of
this particular ALK rearrangement pattern in our patient’s
CTCs is therefore consistent with the observation above
suggesting that these tumors and their CTCs may be
favoring the EMT pathway.
This study further highlights the utility of antibody-
independent microuidic isolation systems for the
isolation and downstream characterization of CTCs
compared with immunomagnetic antibody-dependent
systems. While the numbers of CTCs isolated here are
small and may present substrate limitations to downstream
characterization of CTC, it should be noted that the current
numbers were derived from <2 mL of blood, as opposed to
existing systems which use up to 7.5 mL of blood or more.
In addition, we have previously demonstrated that there is
an association between CTC number and the volume of
blood processed [19]. This suggests a limitation that can
be easily overcome.
The index case presented here raises the possibility
that CTC enumeration based on ALK FISH may be
associated with treatment response with crizotinib by
imaging. The appearance of additional ALK rearrangement
patterns following progression with crizotinib treatment
exhibited a double split in both ALK alleles giving rise to
the 2R2G ALK rearrangement pattern. The additional copy
of the oncogenic ALK may have contributed to disease
progression despite treatment with an ALK inhibitor. This
observation is worthy of further inquiry, because while
the presence of ALK copy number gain is correlated with
crizotinib resistance, as previously reported by Doebele
et al. [40], and in vitro studies have identied potential
resistance mutations in the ALK gene, for example
L1196M, G1269A, S1206Y and G1202R [40, 41], limited
analysis of post-progression biopsies of tumors from a
phase 1 study of LDK378 suggested that these secondary
resistance mutations or gene amplication do not account
for a majority of resistance cases [11]. Hence, further work
with paired re-biopsies and sequential CTC collection may
assist understanding of resistance mechanisms in ALK-
driven tumors.
Conclusions drawn from our study are limited by
its relative small patient population, as was Pallier’s study
[22]. Nonetheless, the converging trend of both studies’
ndings is indicative of the utility and potential of CTCs
as an alternate target of ALK testing in lung cancer and
informs the development of CTC-based technology. More
importantly, these studies provide the basis for subsequent,
large-scale validation studies.
In summary, high concordance of ALK
rearrangement patterns in CTCs and tumors as assessed by
ALK FISH testing indicates that CTCs may have utility as
a non-invasive surrogate diagnostic tool and may be useful
in the longitudinal follow-up for resistance proling. The
availability of a non-invasive tool would improve efforts
to guide ALK-rearranged targeted treatment in NSCLC,
especially in cases without tissue availability. Further
efforts at downstream CTC characterization and culture
following enrichment are ongoing.
MATERIALS AND METHODS
Patient recruitment and blood samples
Patients with conrmed NSCLC were recruited into
this trial. They were naïve for ALK-targeted TKI treatment,
but may have received other forms of chemotherapy. Once
informed consent was secured from these patients, their
blood samples were processed for CTC analysis. The
clinical sample collection protocols were reviewed and
approved by SingHealth Centralised Institutional Review
Board. Clinicopathological information was also recorded
for these patients. Blood samples from healthy donors
were used as controls in this study.

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Histopathology
H&E was done using the Leica ST5010 XL
automated stainer (Leica Biosystems, Wetzlar, Germany)
while periodic PAS-D staining was done using the Ventana
BenchMark Special Stains automated stainer (Ventana
Medical Systems Inc, Tucson, Arizona, USA), following
the respective standard protocols. Histological diagnoses
were made based on the World Health Organization
(WHO) classication [42]. IHC labeling was performed
on the Ventana BenchMark Ultra autostainer (Ventana
Medical Systems Inc, Tucson, Arizona, USA) using
the UltraView detection kit and proprietary Standard
CC1 (SC1) pre-treatment sets. The antibodies used with
their dilution and pre-treatments were as follows: TTF-
1 (Novacastra NCL-TTF-1, clone SPT24, SC1, dilution
1:30), vimentin (DAKO M0725, clone V9, SC1, 1:100)
and E-cadherin (Dako M3612, clone MCH-38, SC1,
1:30) antibodies. Histopathology data was reviewed by
pathologists who had been accredited by the College of
American Pathologists (CAP).
CTC enrichment
Peripheral blood was collected using K2 EDTA
vacutainer® blood collection tube (BD, Singapore)
and processed within 24 hours. Subsequently, 7.5 mL
of whole blood was incubated with red blood cell
(RBC) lysis buffer (G-Biosciences, USA) according
to manufacturer’s recommendations. Lysed RBCs in
the supernatant were discarded after centrifugation.
Remaining cell pellet containing CTCs was resuspended
in ClearCell
®
resuspension buffer prior to CTC enrichment
using the ClearCell® FX system (Clearbridge BioMedics,
Singapore), according to manufacturer’s instruction.
The ClearCell® FX system is an automated CTC
enrichment system driven by the CTChip® FR1, a
microuidic biochip to isolate CTCs based on size,
deformability and inertia. The isolation principle takes
advantage of the inherent Dean vortex ows present in
curvilinear channels for CTC enrichment, termed Dean
Flow Fractionation (DFF) [43]. The enriched CTC sample
output was equally divided into four portions.
Fluorescent in-situ hybridization
Four µm thick FFPE tumor tissue sections were
mounted on positively charged slides and deparafnized.
FISH was subsequently performed using the US
FDA-approved Vysis ALK Break Apart FISH Probe Kit
(Abbott Molecular, Abbott Park, Des Plaines, IL, USA).
The 5’ ALK probe was labeled with SpectrumGreen™
(G) and the 3’ ALK probe with SpectrumOrange™ (R).
ALK FISH for FFPE tissues were considered positive if
at least 15 % of the tumor cells showed abnormal break
apart signals as detailed in the IVD Vysis ALK Break
Apart FISH Probe Kit and by Camidge et al. [33]. A cell is
interpreted as having a split pattern (ALK-positive) when
the 5’ (G) and 3’ (R) signals are separated by two or more
signal diameters. Cells lacking both uorescent signals
were not evaluated.
One portion (one-quarter) of the enriched CTCs
was xed in Shandon CytoSpinTM Collection Fluid
(ThermoFisher Scientic, USA) overnight at 4°C. The
sample was deposited onto positively charged glass slides
by cytospin (800 rpm, 5 mins). All the cells on the slides
were analyzed for the ALK break-apart signal at 1000X
magnication. The scorers analyzing the ALK break-apart
signal on CTCs were blinded to the ALK rearrangement
patterns on the tumor samples, as well as whether the cell
isolated was from patients or healthy controls.
The remaining three portions of the enriched CTCs
were stored at 4°C under validated conditions for future
molecular testing.
Data analysis
Statistical analyses of the data were performed in
GraphPad Prism version 5.00 (GraphPad Software, San
Diego, CA, USA). A non-parametric two-tailed, t-test
(Mann-Whitney) was used for computing statistical
signicances. p value of less than 0.05 were considered
signicant.
ACKNOWLEDGMENTS
The authors wish to acknowledge Clearbridge
BioMedics, Singapore for providing the CTC capturing
technology and technical support. The authors would also
like to express sincere gratitude to patients and healthy
donors for participating in this study.
FUNDING
This study was supported by National Medical
Research Council grants (CSA040/2012 and CS-
IRG1225/2009).
CONFLICTS OF INTEREST
Chwee Teck Lim is an advisor with Clearbridge
Biomedics, Singapore. Man Chun Leong is an employee
of Clearbridge Biomedics, Singapore. All other authors
declare that they do not have any conicts.

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Editorial note
This paper has been accepted based in part on peer-
review conducted by another journal and the authors’
response and revisions as well as expedited peer-review
in Oncotarget.
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