Circulating tumor microemboli (CTM) and vimentin+ circulating tumor cells (CTCs) detected by a size-based platform predict worse prognosis in advanced colorectal cancer patients during chemotherapy
Zhang et al. Cancer Cell Int
Circulating tumor microemboli (CTM) and vimentin circulating tumor cells (CTCs) + detected by a size-based platform predict worse prognosis in advanced colorectal cancer patients during chemotherapy
Dejun Zhang 0 2
Lei Zhao 0 2
Pengfei Zhou 1
Hong Ma 0 2
Fang Huang 0 2
Min Jin 0 2
Xiaomeng Dai 0 2
Xiumei Zheng 0 2
Shaoyi Huang 1
Tao Zhang 0 2
0 Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan 430022, Hubei , People's Republic of China
1 Wuhan YZY Medical Science & Technology Co., Ltd. , Wuhan 430075, Hubei , People's Republic of China
2 Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan 430022, Hubei , People's Republic of China
Background: Circulating tumor cells (CTCs) detected in peripheral blood (PB) of cancer patients can be identified as isolated CTCs and circulating tumor microemboli (CTM). This study aimed to evaluate the prognostic value of CTM detection and CTC phenotype in advanced colorectal cancer (CRC) patients during chemotherapy. Methods: A size-based platform for CTC isolation was applied. PB samples (5 ml) from 98 advanced CRC patients during 2-6 cycles chemotherapy were collected for CTC detection, and CTC count was correlated to patient's clinicopathological characteristics and clinical outcome. And CTC phenotype was measured by immunofluorescent staining and evaluate the predictive significance on survival in 32 CTCs-positive patients with advanced CRC. Results: Forty-eight of 98 patients were CTCs-positive, including 18 CTM-positive patients, and CTC detection was positively correlated with lymphatic invasion (P = 0.049), TNM stage (P = 0.023), and serum CEA level (P = 0.014). Moreover, Kaplan-Meier survival and Cox regression analyses revealed that the presence of CTCs was an independent factor for poor PFS and OS (P < 0.05) in advanced CRC patients during chemotherapy, and CTM-positive patients had shooter survival than isolated CTCs-positive patients (P < 0.05). Furthermore, patients with vimentin+ isolated CTCs/ CTM had shorter PFS and OS compared with CK+ CTCs (P < 0.05). Conclusions: This study provided evidence that the presence of CTCs was positively correlated with poor prognosis, and furthermore, CTM and vimentin+ CTCs predicted poorer survival, which indicated that CTM and vimentin+ CTCs detected by a sensitive platform could be used to improve prognostic value of CTCs in advanced CRC patients under treatment.
Circulating tumor cells; Circulating tumor microemboli; Colorectal cancer; Survival; Vimentin
Colorectal cancer (CRC) is the third most common
cancer in male and the second most common in female
worldwide, and contributes the fourth cause of cancer
death in male and the third in female . For advanced
CRC patients, although many patients benefit from
chemotherapy to some extent, for some patients
excessive chemotherapy was unnecessary due to inefficiency,
moreover, multiple adverse effects seriously lower their
life quality . Therefore, new prognostic factors which
could be used to identify patients who would benefit
from chemotherapy are needed.
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Circulating tumor cells (CTCs) non-invasively isolated
from peripheral blood can serve as a “liquid biopsy” and
as a source of valuable tumor markers. Many studies
reported that CTC detection had prognostic and
therapeutic significance in CRC [3–7]. Moreover, in advanced
CRC patients, the presence of CTCs before and during
treatment had been proved to be an independent
predictor of progression-free survival (PFS) and overall survival
(OS) [3, 6], and a key factor to improve the accuracy in
assessing the effectiveness of first-line treatment .
However, CTC detection, enumeration and molecular
characterization are quite challenging, because CTCs are
rare in peripheral blood of patients. The Veridex CellSearch
system (Veridex LLC, Raritan, NJ) utilizes magnetic beads
coated by anti-EpCAM antibody to capture cells followed
by the fluorescence staining to identify CTCs, defined as
CK8/18/19+/DAPI+/CD45− cells . However, EpCAM
expression is dependent on the local microenvironment
and is down-regulated in disseminated cells .
Epithelialmesenchymal transition (EMT) of tumor cells is induced in
the bloodstream , which leads to mesenchymal tumor
cells with stem-like phenotype [11, 12], and loss of
epithelial phenotype . This is quite probably the reason why
the CTC detection rates and counts in the CellSearch
system are generally low. For example, 17 of 66 non-metastatic
CRC patients (26%) had ≥2 CTCs per 7.5 ml peripheral
blood , and in another study, only 19 of 239
preoperative CRC patients (~8%) had ≥1 CTC per 7.5 ml peripheral
blood . Therefore, CTCs as an independent prognostic
marker, need a more sensitive method to further facilitate
the evaluation of CTC detection.
Here, a sensitive size-based platform for CTC
isolation was applied, which could filter the hemocytes with
small diameter and capture the tumor cells with relatively
big diameter, followed by Romanowsky dye and
immunofluorescent staining to identify CTCs. In this study,
peripheral blood samples (5 ml) from 98 advanced CRC
patients during 2–6 cycles chemotherapy were collected
to detect CTCs for Romanowsky dye staining, then CTC
levels were correlated with clinicopathological
characteristics and patient’s survival. Moreover, CTC
phenotype was measured by immunofluorescent staining in 32
CTCs-positive patients with advanced CRC. It was
demonstrated that CTC detection by a size-based platform
was positively correlated with lymphatic invasion, TNM
stage, serum CEA level and poor survival, and CTM and
vimentin+ CTCs predicted poorer survival in advanced
CRC under treatment.
Union Hospital, Huazhong university of science and
technology, from January, 2013 to April, 2013, and
peripheral blood samples from patients were collected.
The TNM classification of CRC was based on
American Joint Committee on Cancer (AJCC) 7th edition.
The clinicopathologic characteristics of patients were
classified according to the chart records, as showed in
This prospective study was double-blinded in terms of
blood draw, CTC detection and identification. For the
purpose of this study, healthy donors were those without
abnormal cells detected by this size-based platform for
CTC isolation in peripheral blood.
The informed consent approved by ethics committee
of Union Hospital, Huazhong university of science and
technology had been obtained from all patients before
examination. All procedures performed in studies
involving human participants were in accordance with the
ethical standards of the ethics committee of Union Hospital,
Huazhong University of science and technology and with
the Helsinki declaration and its later amendments or
comparable ethical standards.
CTC detection by a size‑based platform
The 5 ml blood sample of advanced CRC patient was
diluted up to 8 ml with 0.9% physiological saline
containing 0.2% paraformaldehyde, then measured on an
automated testing platform following manufacturer’s
instructions, as described in an earlier study by Vona
et al. . This platform was composed of a membrane
with 8 μm size pores and a automated testing device.
The captured cells including abnormal cells and
residual haemocytes on the membrane were stained with
Romanowsky dye (eosin and methylene blue) and
immunofluorescent staining. The candidate CTCs were
identified independently by 3 senior cytopathologists.
The captured tumor cells on the membrane were
processed with Cytofix/Cytoperm Fixation/Permeabilization
solution (BD, New Jersey, USA) for 10–15 min, incubated
with 10% Goat Serum (Jackson, West Grove, USA) for
30 min at room temperature, then incubated with
antiCK8/18/19, anti-vimentin (Abcam Trading (Shanghai)
Company Ltd., Shanghai, China) and anti-CD45 (Santa,
Texas, USA) antibody overnight at 4 °C. The next day
they were incubated with secondary antibodies, Alexa
Fluor 488-conjugated goat anti-mouse, Alexa Fluor
546-conjugated goat anti-rabbit, Cy5-conjugated goat
anti-rabbit (InvitrogenTM, Thermo Fisher Scientific,
Waltham, USA), and Hoechst (SIGMA, St. Louis, MO)
for 1 h at room temperature. Then they were imaged by
Table 1 Relationship between circulating tumor cells
(CTCs) and clinicopathological characteristics in advanced
No. of patients (%) CTCs
48 (49.0) 50 (51.0)
All data were analyzed using SPSS 16.0 statistic software
(SPSS Inc., Chicago, IL, USA). The associations between
CTCs and clinicopathologic variables were evaluated
with χ2 tests. Survival curves were calculated using the
Kaplan–Meier method. Factors of prognostic significance
were investigated with the univariate and multivariate
Cox regression model. For all tests, the P ≤ 0.05 indicated
Abnormal cells detected by a size‑based platform for CTC
isolation in peripheral blood of patients with advanced
In this study a size-based platform for CTC isolation was
applied. This platform was mainly composed of a filter
membrane with 8 μm size pores and an automated testing
device. A spiking test was conducted to test the capture
efficiency and sensitivity of this platform, in which HT29
colorectal cancer cells were added into 5 ml peripheral
blood of healthy donors. the transparent membrane in
the filter got a clear background after CTC isolation and
Romanowsky staining, which facilitated the procedure of
indentifying CTCs and CTC phenotype (Fig. 1a, b). The
results showed that this method for isolating CTCs was
reliable and robust (Fig. 1c, d).
Based on the criteria proposed by other researchers
[16–18] and our own experience, there were 6 criteria of
cell morphological characteristics for evaluating
abnormal cells captured in peripheral blood: (1) the nuclear
atypia: irregularity of nuclear shape, may be nodular or
lobulated etc.; (2) a high nuclear–cytoplasmic ratio: >0.8;
(3) a large cell diameter (the long diameter): >15 μm;
(4) the hyperchromatic nuclei were dyed unevenly (due
to the increase of chromatin and the thicker particles in
cancer cells, the nucleus was hyperchromatic); (5) the
thickened nuclear membrane was sunken, wrinkled and
jagged; (6) the nuclear chromatin margination (nucleus
side-shift), or a large nucleoli, or abnormal nuclear
Abnormal cells captured by this method were
identified as CTCs in colorectal cancer, only if they met no
less than 4 criteria above, or met the 6th criterion and
any other 2 criteria (Fig. 1e, f ). If they met any 3
criteria except the 6th criterion, or met only the 6th criterion,
they were identified as the suspected CTCs (Fig. 1g, h).
Besides, CTC cluster composed of three or more CTCs
was recognized as circulating tumor microemboli (CTM)
(Fig. 1i, j), while other cell clusters were recognized as
the suspected CTM. However, some cells should not be
present in peripheral blood normally (e.g. epithelial cells,
endothelial cells) (Fig. 1k, l), or were of undetermined
origin, all those cells were regarded as non-blood cells.
The relationship between CTCs/CTM
and clinicopathological characteristics in advanced CRC
In this study, ninety-eight advanced CRC patients
during 2–6 cycles chemotherapy were subjected to CTC
Fig. 1 Abnormal cells detected in peripheral blood (PB) of advanced CRC patients. a The clear background of a membrane in the filter after
Romanowsky staining. b The spiking HT29 cells captured by the size-based platform for CTC isolation (as indicated by the black arrows). c The
capture efficiency of cancer cell linces HT29, SKBR-3 and A549. d The sensitivity of isolating HT29 cells. e, f The single CTC (as indicated by the red
arrows) detected in PB. g, h The suspected CTC (as indicated by the yellow arrows) in PB. i, j CTM (as indicated by the red arrows) detected in PB. k
Epithelial cells (as indicated by the green arrows) detected in PB. l Endothelial cells (as indicated by the green arrows) detected in PB (a, ×10
magnification; b, c, ×60 magnification; f–m, ×100 magnification)
isolation and enumeration, forty-eight patients were
CTCs-positive, including 18 CTM-positive patients. The
association of CTCs with the clinicopathological variables
of patients was shown in Table 1. CTCs were positively
correlated with tumor de-differentiation (P = 0.004),
lymphatic invasion (P = 0.049), TNM stage (P = 0.023), and
serum CEA level (P = 0.014). By contrast, no significant
association was found between CTCs-positive and other
clinicopathological characteristics (P > 0.05 for all others),
such as gender, age, tumor size, tumor location, serum
CA199 level, and depth of invasion (Table 1). Serum CEA
levels in CTCs-positive patients were higher than
CTCsnegative patients (334.8 ± 194.7 vs. 115.6 ± 71.43 ng/ml,
P = 0.0155) (Fig. 2a), while there was no statistical
significance in serum CA199 levels between CTCs-positive and
CTCs-negative patients (1486 ± 498.7 vs. 651.1 ± 339.2
U/ml, P = 0.0887) (Fig. 2b).
Furthermore, CTC enumeration of all 98 advanced
CRC patients ranged from 0 to 195 (mean ± SE:
9.663 ± 2.775), and CTM enumeration ranged from 0 to
17. And CTC enumeration was increasing with decreased
tumor de-differentiation (poor vs. middle, P = 0.0191;
poor vs. high, P = 0.0359), increased lymphatic invasion
(N2b vs. N0, P = 0.0429; N2b vs. N1, P = 0.0361; N2b
vs. N2a, P = 0.1037), TNM stage (IVb vs. III, P = 0.0186;
IVb vs. IVa, P = 0.1019) and serum CEA level (CEA > 10
vs. CEA ≤ 10 ng/ml, P = 0.0026) (Fig. 2c–g).
CTCs/CTM predicted poor survival in advanced CRC
patients under treatment
Based on univariate Cox regression analyses for all
factors (Table 2), CTCs (P < 0.0001), lymphatic invasion
(P = 0.042), TNM stage (P < 0.001), and high CEA level
(P = 0.0027) were closely related with PFS. The
multivariate Cox regression model further demonstrated that
CTCs (P = 0.015) and TNM stage (P = 0.013) were
independent prognostic factors for shorter PFS (Table 2). And
the Kaplan–Meier survival curves showed that
CTCspositive patients with advanced CRC had a significantly
unfavorable PFS (9 vs. 17 months, P = 0.0006) (Fig. 3a),
and furthermore, CTM-positive patients had shorter
PFS than CTCs-positive patients (6 vs. 12 months,
P = 0.0052) (Fig. 3c).
Moreover, based on univariate Cox regression
analyses for all factors (Table 2), CTCs (P = 0.048), lymphatic
invasion (P < 0.001), and TNM stage (P = 0.015) were
closely related with poor OS. Although the
multivariate Cox regression model demonstrated that lymphatic
invasion (P < 0.001) and TNM stage (P = 0.017) were
independent prognostic factors for PFS but not CTCs
(Table 2), the Kaplan–Meier survival curves showed that
CTCs-positive patients with advanced CRC had a
significantly unfavorable OS (16.5 vs. 23 months, P = 0.0278)
(Fig. 3b), and CTM-positive patients had worse OS than
CTCs-positive patients (12 vs. 18 months, P = 0.0228)
Vimentin+ isolated CTCs/CTM predicted worse survival
in advanced CRC patients under treatment
Thirty-two CTCs-positive patients were subjected to
CTC isolation again to identify CTC phenotype by
immunofluorescence. The samples were stained with
anti-CK8/18/19 antibody (epithelial marker),
anti-vimentin antibody (mesenchymal marker), anti-CD45 antibody
(for leukocytes), and hoechst (for nucleus). In this study,
four CTC phenotypes were detected: CK+/Vimentin+/
CD45− CTM (Fig. 4a), CK−/Vimentin+/CD45− CTM
(Fig. 4b), CK−/Vimentin+/CD45− isolated CTCs
(Fig. 4c), and CK+/Vimentin−/CD45− isolated CTCs
(Fig. 4d). For further analysis, 13 patients with vimentin+
CTCs/CTM (CK+/Vimentin+/CD45− CTM, CK−/
Vimentin+/CD45− CTM, CK−/Vimentin+/CD45−
isolated CTCs) and 19 patients with CK+ CTCs (CK+/
Vimentin−/CD45− isolated CTCs) were identified.
Interesting, it was found that all of CTM (detected in 11
of 11 patients) were vimentin-positive, while most of the
isolated CTCs (detected in 19 of 21 patients) were
CKpositive. Moreover, the Kaplan–Meier survival curves
showed that advanced CRC patients with vimentin+
CTCs had significantly shorter PFS and OS compared
with CK+ CTCs (6 vs. 11 months, P = 0.0314; 11 vs.
20 months, P = 0.0147) (Fig. 4e, f ).
CTC detection in peripheral blood was recognized as
“liquid biopsy” in solid tumors, because it could be
performed easily, frequently, and less invasively [19, 20].
There was increasing evidence which prove CTCs as
the clinical marker for diagnostic, prognostic, and
pharmacologic purposes [21, 22]. Hence, CTC detection and
characterization had become a research focus worldwide.
Although many studies about CTCs proved that high
baseline CTC count was positively correlated with worse
prognosis in colorectal cancer by CellSearch system [6,
23, 24], the CTC detection rate and count in CellSearch
system were generally low, and many approaches of CTC
isolation had been developed recently. In this study, we
applied a size-based platform for CTC isolation, and the
spiking tests showed the capture efficiency and
sensitivity of this platform was reliable and robust. Moreover,
the CTC detection rate in advanced CRC patients during
2 ~ 6 cycles chemotherapy was 49% (48 of 98 patients),
which was significantly higher than that detected by
CellSearch system (data showed in meta-analysis) [23, 24],
and it was consistent with the results of another study
which compared CTC detection rate of the size-based
Fig. 2 The relationship between CTCs/CTM and clinicopathological characteristics in advanced CRC. a Serum CEA levels in CTC-positive patients
were higher than CTC-negative patients (P = 0.0155). b There was no statistical significance in serum CA199 levels between CTC-positive and
CTCnegative patients (P = 0.0887). c The correlation of CTC count with tumor de-differentiation (poor vs. middle, P = 0.0191; poor vs. high, P = 0.0359).
d CTC count of patients with depth of invasion (T4a vs. T4b, P = 0.7826; T4a vs. T3, P = 0.3708; T4a vs. T1 + T2, P = 0.4762). e The correlation of CTC
count with lymphatic invasion (N2b vs. N0, P = 0.0429; N2b vs. N1, P = 0.0361; N2b vs. N2a, P = 0.1037). f The correlation of CTC count with TNM
stage (IVb vs. III, P = 0.0186; IVb vs. IVa, P = 0.1019). g CTC count of patients with CEA > 10 pg/ml was more than CEA ≤ 10 pg/ml (P = 0.0026)
platform and the CellSearch system in esophageal
carcinoma . The high sensitivity of this size-based
platform could be mainly attributed to two factors: Firstly,
the CellSearch system only regarded tumor cells with
epithelial phenotype in peripheral blood as CTCs, which did
not take other properties and processes which were
associated with malignant potential into consideration, such
as EMT, cohesive and collective cell migration .
Secondly, this size-based platform captured malignant cells
by the difference of diameter and deformability between
abnormal cells and haemocytes, hence it could isolate
more abnormal cells for further identifying CTCs.
However, when comparing the CTC detection rates by ISET
(isolation by size of epithelial tumor cells) in some studies
[26–29], there was a subtle difference in this study. The
discrepancy might due to the heterogeneity of different
cancers, different stages of tumor, and whether
undergoing treatment or not, etc.
Table 2 Univariate and multivariate analysis of prognostic factors for progression-free survival (PFS) and overall survival
(OS) in advanced colorectal cancer
We also observed the relationship between CTCs and
clinicopathological characteristics, as shown in Table 1.
It was found that CTCs were associated with tumor
dedifferentiation, lymphatic invasion, TNM stage, and
serum CEA level, which were consistent with the results
of previous studies [30, 31]. In addition, serum CEA
values in CTCs-positive patients were higher than
CTCsnegative patients, which indicated that patients with high
CEA levels had more opportunities to be CTCs-positive.
Moreover, CTC count was increasing with decreasing
tumor de-differentiation, increasing lymphatic invasion,
TNM stage, and serum CEA level. Therefore, although
the decisions on stage of disease still did not include the
results of CTC assessment, the presence of CTCs might
be an adjunct to staging , and it could be expected
that CTC detection predicted the properties and
processes of the disease (e.g. lymphatic invasion, TNM stage,
and serum CEA level).
This study found that the presence of CTCs was
associated with decreased survival in advanced CRC patients
with 2–6 cycles chemotherapy, and Cox regression analyses
showed that CTC detection was an independent
prognostic factor for survival, which was consistent with previous
studies [23, 24, 33, 34]. Notably, it was reported that the
Fig. 3 The relationship between CTCs/CTM and PFS/OS in advanced CRC. a, b The PFS and OS of CTC-positive patients were shorter than
CTC-negative patients (P = 0.0006, P = 0.0278). c, d The PFS and OS of CTC-positive patients were worse than CTM-positive patients (P = 0.0052, P = 0.0228)
relationship between CTC detection and prognosis was
more significant and convincing when the blood samples
were collected during treatment than at baseline [23, 24],
which indicated that sample collection during treatment
was preferable for CTC detection to predict CRC patient’s
outcomes. That was the reason why we recruited the
advanced CRC patients with 2–6 cycles chemotherapy in
this study. Moreover, CTM was captured by this size-based
platform, and CTM-positive patients with advanced CRC
had worse survival than isolated CTCs-positive patients. It
was reported that tumor cells within CTM could be
protected from anoikis and were relatively resistant to
cytotoxic drugs , and CTM was an independent prognostic
factor [35, 36]. Hence, CTM would be more malignant and
aggressive than isolated CTCs.
CTCs were comprised of heterogeneous cells including
epithelial tumor cells, tumor cells undergoing EMT and
tumor stem cells etc. [12, 37, 38], and circulating
epithelial tumor cells had been shown to respond to therapy
in the same way as the primary tumor , while the
detection of EMT markers (LOXL3 and ZEB2) for CTCs
in mCRC predicted poor survival and therapy response
during treatment , hence CTC molecular
characterization could offer the potential to better understand
the biology of metastasis and resistance to established
therapies . In this study CTC phenotype was
measured by immunofluorescent staining for CK8/18/19
(epithelial marker) and vimentin (mesenchymal marker), and
it was found that all CTM were vimentin-positive, while
most of the isolated CTCs were CK-positive. Moreover,
patients with vimentin+ CTCs had worse survival than
CK+ CTCs. To our knowledge, this was the first study
that evaluated the prognostic role of CTCs with epithelial
and mesenchymal phenotype in advanced CRC patients
In this study, it was found that the presence of CTCs was
associated with decreased survival, and was an
independent prognostic factor for outcome in advanced CRC
patients during chemotherapy. Moreover, patients with
CTM had shorter survival than those with isolated CTCs,
and patients with vimentin+ CTCs had worse survival
compared to those with CK+ CTCs. Therefore, this study
Fig. 4 The relationship between vimentin+ CTCs and PFS/OS in advanced CRC. The captured tumor cells were stained with anti-CK8/18/19
antibody for epithelial marker (green fluorescence), anti-vimentin antibody for mesenchymal marker (yellow fluorescence), anti-CD45 antibody for
leukocytes (red fluorescence), and hoechst for nucleus (blue fluorescence). The CTM detected in peripheral blood of patients were CK+/Vimentin+/
CD45− (a) or CK−/Vimentin+/CD45− (b) phenotype. The isolated CTCs were CK−/Vimentin+/CD45− (c) and CK+/Vimentin−/CD45− (d)
phenotype. e, f Patients with vimentin+ CTCs had worse PFS/OS compared with CK+ CTCs (P = 0.0314, P = 0.0147)
had demonstrated that CTM and vimentin+ CTCs could
be used to improve prognostic value of CTCs in advanced
CRC patients under treatment.
AJCC: American joint committee on cancer staging; CTCs: circulating tumor
cells; CTM: circulating tumor microemboli; CA125: carbohydrate antigen 125;
CEA: carcinoembryonic antigen; CK: cytokeratin; CRC: colorectal cancer; EMT:
epithelial to mesenchymal transition; OS: overall survival; PFS: progression-free
survival; PB: peripheral blood; TNM: tumor-node-metastasis.
ZDJ and ZL carried out CTC detection and immunofluorescent staining,
drafted the manuscript, and participated in the design of the study. ZPF, MH
and HSY carried out the identification of candidate CTCs independently.
DXM and ZXM collected the clinicopathologic variables of patients. HF and
JM performed the statistical analysis and helped to draft the manuscript. ZT
conceived of the study, and participated in its design and coordination. All
authors read and approved the final manuscript.
The authors acknowledged Dr. Congli Cai, Ting Ye, Peng Xu for their technical
assistance in Wuhan YZY Medical Science & Technology Co., Ltd.
The authors declare that they have no competing interest.
All procedures performed in studies involving human participants were in
accordance with the ethical standards of the ethics committee of Union
Hospital, Huazhong University of science and technology and with the 1964
Helsinki declaration and its later amendments or comparable ethical standards.
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