Intradermal microbubbles and contrast-enhanced ultrasound (CEUS) is a feasible approach for sentinel lymph node identification in early-stage breast cancer
Xie et al. World Journal of Surgical Oncology
Intradermal microbubbles and contrast- enhanced ultrasound (CEUS) is a feasible approach for sentinel lymph node identification in early-stage breast cancer
Fei Xie 0 3
Dongjie Zhang 0 3
Lin Cheng 0 3
Lei Yu 2
Li Yang 2
Fuzhong Tong 0 3
Hongjun Liu 0 3
Shu Wang 2
Shan Wang 1
0 Department of Breast Disease, Peking University People's Hospital , Beijing , China
1 Department of Gastrointestinal Surgery, Peking University People's Hospital , Beijing , China
2 Department of Ultrasound Diagnosis, Peking University People's Hospital , Beijing , China
3 Department of Breast Disease, Peking University People's Hospital , Beijing , China
Background: Microbubbles and contrast-enhanced ultrasound (CEUS) is a new technique for locating sentinel lymph node (SLN). The aim of this study is to explore the feasibility of SLNs tracing by CEUS using microbubbles in breast cancer patients and the value of enhancing patterns in diagnosing lymph nodes metastases. Methods: A clinical trial was registered (trial registration: ChiCTR-DDT-13003778). One hundred and one consecutive consenting patients with breast cancer undergoing SLN biopsy were enrolled. Before the surgery, microbubble was injected periareolarly. Lymphatic drainage pathway was detected by CEUS, and guidewire was deployed to locate the SLN before the operation. Blue dye was also used to help in tracing sentinel lymph node during the operation. The identification rate and the accuracy rate were recorded. Enhancing patterns of lymph nodes were recorded and compared with the pathological diagnosis. Results: Of the 101 cases, SLNs in 99 cases were successfully identified by at least one tracer, including 98 cases identified by CEUS and 97 cases by blue dye. There was no significant difference between the two methods (P = 0.705). Guidewires were deployed successfully in all 98 cases, and the localized SLNs were all isolated successfully in the following operations. The status of SLNs isolated by CEUS was completely identical to that of the whole axillary lymph node while 7.1 % cases were misdiagnosed as negative by blue dye method. The sensitivity of predicting SLNs metastases by CEUS enhancing pattern was 81.8 %, the specificity was 86.2 %, and the positive and negative predictive values were 75.0 and 90.3 %, respectively. Conclusions: Microbubbles and CEUS are feasible approaches for SLN identification. The enhancing patterns on CEUS may be helpful to recognize the metastasizing SLNs. This novel method may be a promising technique for the clinical application.
Sentinel lymph node (SLN); Sentinel lymph node biopsy (SLNB); Microbubbles; Contrast-enhanced ultrasonography (CEUS); Blue dye; SonoVue; Breast cancer
Axillary lymph node status is an important prognostic
factor in patients with breast cancer [
]. Sentinel lymph
node (SLN) biopsy has been the standard procedure for
axillary staging in early breast cancer with clinical
normal nodes [
]. Non-sentinel lymph node (NSLN)
metastasis is detected in 35–50 % of SLN positive patients
]. Although a number of studies have investigated
models or nomograms for non-sentinel lymph node
(NSLN) metastasis prediction, none of them can predict
the probability of NSLN metastasis correctly [
Hence, if any SLN is positive, the standard procedure
remains axillary lymph node dissection (ALND).
The definition of sentinel lymph nodes is the initial
lymph nodes that receive lymphatic drainage from the
primary tumor. Some markers are used to label the
sentinel lymph nodes along the lymphatic drainage pathway.
The traditional method for sentinel lymph nodes tracing
is a dual-labeled technique involving blue dye and
radiolabeled colloid [
]. The identification rate of the
duallabeled technique has been shown to be up to 96 %, with
false negative rates between 5 and 10 % [
Nevertheless, the half-life of the technetium-99m (99mTc) is
up to 6 h, while molybdenum-99 (99Mo) decays rapidly,
which may restrict the scheduling of radioisotopes
production and delivering. Handling and disposal of
radioisotopes are also severe challenges for hospital
management. All these drawbacks have limited the
widespread use of radioisotopes for SLN biopsy.
Isosulfan blue, patent blue, and methylene blue are dye
tracers that are frequently used. The identification
rates of SLN biopsy by blue dye alone range from 66
to 94 %, with false negative rates of 0 to 12 % [
Considering the acceptable accuracy and absence of
radioactive contamination, the blue dye has become the
alternative standard control tracer in many studies [
Poor tissue penetration is the obvious drawback of blue
dye technique. Surgeons have to anatomize the lymphatic
vessels cautiously to find the SLN during the procedure.
The constraints of the existing SLN biopsy techniques
have led to the development of alternative methods.
Indocyanine green fluorescence (ICG) [
superparamagnetic iron oxide nanoparticles (SPIO) [
and contrast-enhanced ultrasound (CEUS) using
microbubbles have been reported as new feasible alternative
techniques for the SLN biopsy in breast cancer [
Sulfur hexafluoride microbubbles is the most popular
ultrasound contrast agent at present, which plays a role
as an inert gas in vivo. There is no evidence of
metabolism of sulfur hexafluoride. Furthermore, diameters of
the bubbles range from 2–10 μm (mean 2.5 μm), which
are much less than those of the red blood cells (mean
7.2 μm). So the bubbles can easily pass through the
blood capillary and lymphatic microvessels, even across
the alveolar epithelium. Therefore, the bubbles also can
be cleared up by the lung and kidney easily. On the
other hand, there are no iodine and proteins in sulfur
hexafluoride microbubbles, which prevents a great
portion of patients from having allergy. Sulfur hexafluoride
microbubbles may be a promising SLN marker
considering its safety and reliability. Nevertheless, there
are very few published studies of sentinel lymph node
biopsy done by CEUS using microbubbles in human
The aim of this study is to validate the effectiveness of
the identification of SLNs using microbubbles and CEUS
in patients with early breast cancer. Also, we attempted
to explore the potential value of CEUS in the
preoperative diagnosis of SLN metastases in patients with
early breast cancer.
The study was designed as self-control and registered at
Chinese Clinical Trial Registry (the registration number
is ChiCTR-DDT-13003778). The study was approved by
the institutional ethics committee of Peking University
People’s Hospital (IRB approval number is 2012045).
Informed consent was obtained from all enrolled patients
(the version number is modified revision 2012.12.18).
Between October 2013 and October 2014, 101
consecutive consenting patients with early-stage breast cancer
who were scheduled for SLN biopsy were recruited into
the study. Exclusion criteria included inflammatory
breast cancer, previous surgical approach in the
ipsilateral axillary region, history of sulfur hexafluoride allergy,
axillary lymph node involvement proved by histology or
cytology, and stage IV disease. Patients whose axillary
lymph nodes were suspiciously positive accept
fineneedle aspiration cytology (FNAC) before the operation.
If the pathology result was negative, there would still be
an indication for SLN biopsy. Patients receiving
neoadjuvant therapy were not permitted. All the patients were
female and treated in Peking University People’s
Hospital. Their ages ranged from 22 to 82, and the median
age was 54. In the 101 patients, 49 (48.5 %) had the
primary breast lesion located on left side, and 52 (51.5 %)
had right breast lesions. Core-needle biopsy (CNB) was
performed in 24 cases to confirm the breast cancer;
FNAC was performed in the remaining 77 cases. Patients
diagnosed by FNAC received a frozen pathological
diagnosis during the procedure. As for breast treatment, 36
patients underwent breast-conserving surgery, and 65
patients received total mastectomy (Table 1).
Instruments and materials
All sonographic examinations were performed by two
experienced sonographer using two uniform GE Logiq
E9 scanners (GE Healthcare) equipped with high-frequency
linear array probes (ML6–15-D) and contrast pulse
sequences (CPS). Microbubbles composed of
phospholipidstabilized membrane and sulfur hexafluoride gas in the
inside (SonoVue, Bracco Imaging) were used as ultrasound
contrast agent. SonoVue presented as a dry powder
and needed to be prepared by 5 mL of normal saline
into 8 μl/ml sulfur hexafluoride microbubbles. The
ampoule was shaken vigorously more than 30 s every
time before injection to ensure a homogeneous
suspension of bubbles. Methylene blue (Jumpcan
Pharmaceuticals) was used as a blue dye. A 7-cm-long double hook
guidewire (20G, LW0077, BARD) was used to locate the
sentinel lymph nodes.
Before the SLN biopsy, patients received a gray-scale
ultrasound examination of the axilla first, so the
sonographers had a general idea of the position of lymph
nodes. Then 1.5-mL microbubble suspension was
intradermally injected into the upper lateral areola. If an
incision biopsy was performed in upper outer quadrant
previously, we chose the outer edge of the incision as
the injection site instead. After injection, subcutaneous
lymphatic drainage pathway directing to the axilla could
be detected immediately on contrast pulse sequencing in
most cases. Moving the probe along the lymphatic
channels, the areas of contrast agent accumulation could be
detected, which were the enhanced lymph nodes. Usually,
the passing time from injection to axillary node mapping
was between 5 and 67 s. Contrast agent remained in the
SLN for up to 4 min. Massaging the areola for 10 s
intensified the image again. If the lymphatic vessel was not
detected successfully in a few cases, another injection was
given. If no obvious lymphatic filling was obtained or no
lymph node developed after two consecutive injections,
the case was recorded as a failure. The conventional
grayscale window was alive synchronously to confirm the
presence of an architecturally defined SLN. In some cases,
an apparently enhanced lymph node supposed to be
an SLN on contrast pulse sequencing was difficult to
recognize in a gray-scale pattern.
Once identified, contrast-enhanced ultrasound-identified
SLNs were localized with double hook guidewire. After
insertion, the tails of the guidewire were covered with a
sterile dressing. Considering the unavoidable frequent
movements of an upper limb, and the discomfort caused
by a foreign matter in the axilla, it was more appropriate to
perform this procedure just before the SLN biopsy.
Immediately after the induction of general anesthesia,
1-mL blue dye was intradermally injected into the
periareolar upper outer quadrant region. If an incision
biopsy was performed in upper outer quadrant
previously, the outer edge of the incision was taken to be
the alternative injection site. Five minutes later,
standard SLN biopsies were performed by four well-trained
surgeons, respectively. All blue dye-containing lymph
nodes, guidewire-directing lymph nodes, and any
suspicious lymph nodes during the exploration were excised
and numbered in accordance with the following
instructions: both blue dye- and guidewire-containing nodes
(bub+/dye+) were labeled “1”; only blue dye-containing
nodes were labeled (bub-/dye+) “2”, “3” for only
guidewire-containing nodes (bub+/dye-), and “4” for
neither blue dye- nor guidewire-containing nodes
(bub-/dye). All the lymph nodes were sent for histologic analysis.
Standard axillary lymph node dissection was performed if
any of the nodes proved positive.
The identification rate was defined by the proportion
of patients with SLNs identified with either method.
Data were subjected to χ2 test or Fisher’s exact test
using SPSS statistical software version 17.0 (SPSS Inc.
Chicago, IL, USA). The level of significance was set
at P < 0.05.
CEUS technical success
In 98 of 101 cases, lymphatic channels draining from the
primary lesion and at least one lymph node were clearly
visualized. In the remaining 3 cases, lymphatic vessels
were visualized clearly but no lymph node was
visualized. These 3 cases had stage III disease, and tumor
thrombus in capillaries was proved by pathological
diagnoses. In the 98 lymph nodes visible cases, only one
SLN was detected on CEUS in 84 cases (85.7 %), 11
cases (11.2 %) had two sequentially visualized SLNs, and
3 cases (3.0 %) had three ordinally enhancing SLNs
(Fig. 1). The first one or two enhanced nodes were
deployed guidewire, respectively. The procedure was
successful in all the 98 cases, and SLNs marked by
guidewires were isolated correctly in the following
operations (Fig. 2). There was no displacement of wires,
hematoma, or infections. It was worth noting that it is
better to perform the insertion just before the surgery.
Of the 101 cases, SLNs in 99 cases were successfully
identified by at least one tracer, including 98 cases
identified by CEUS and 97 cases by blue dye. The
identification rates of CEUS, blue dye, and the combined method
were 97.03 % (98/101), 96.04 % (97/101), and 98.02 %
(99/101), respectively (P = 0.705). In total, 271 SLNs were
excised. CEUS method detected less SLNs than blue dye
method (115 vs. 211). While the positive rates of SLNs
identified by CEUS was 35.7 % (41/115), significantly
higher than that by blue dye (21.8 %, 46/211) (P = 0.001)
Axillary lymph node involvement was confirmed in 36
cases by paraffin pathology while ALND was performed
in 41 cases. The other 5 cases received ALND because
suspicious positive lymph nodes were found during the
sentinel lymph node biopsy (SLNB), although the frozen
pathology was negative. Among the 36 axillary involved
cases, SLNs in 33 cases were successfully identified by
CEUS and all were diagnosed positive by frozen
pathology; the other 3 cases had stage III disease and tumor
thrombus in the capillaries so the CEUS method was a
failure; SLNs in 34 cases were successfully identified by
blue dye, but 1 case was misdiagnosed as negative (the
positive SLN was labeled CEUS+/blue dye−).
Furthermore, SLN identification by CEUS was independent of
disease stage and molecular subtype (Table 3).
Pattern of CEUS enhancement
The pattern of enhancement of SLNs was classed into
three types: type I, SLN was obviously and
homogeneously enhanced; type II, SLN was obviously enhanced,
but the enhancement was not homogeneous, with
hypoperfusion or non-perfusion area present; type III, SLN
was weakly enhanced or not enhanced (Fig 3).
Comparing the enhancement pattern with paraffin pathological
diagnosis of SLNs, we found that type I was the most
typical pattern in non-involved SLNs (56/65), while type
II enhancement was more common in involved SLNs
(23/33) (P < 0.001). If we considered the SLNs with type
II or III enhancement as positive nodes and type I as
negative, the sensitivity of CEUS in the diagnosis of
SLNs metastases was 81.8 % (27/33), the specificity was
86.2 % (56/65), and accuracy rate was 84.7 % (83/98). The
positive predictive value was 75.0 % (27/36); the negative
predictive value was 90.3 % (56/62) (Table 4).
SLNB has become the routine procedure in patients
with early breast cancer, and ALND is still a standard
treatment for SLN involved case. Although according to
the ACOSOG Z0011 and IBCSG 23-01 data, patients
with limited SLN metastatic breast cancer treated with
breast conservation and systemic therapy may omit
]. The conclusions still need to be
validated by more well-designed trials. So the primary value
of SLNB remains consisting in indicating axillary status
with minimal trauma. The combination of radioisotopes
and blue dye is a popular method for SLN tracing.
However, the limitations are not negligible. The diameter of
radioactive colloid is usually between 50 and 100 nm,
while the endothelial gap of subcutaneous lymph vessel
is between 120 and 500 nm; so the radioactive colloid
cannot pass through the lymph–vessel freely but can be
absorbed by endocytosis. Therefore, many hours are
needed to detect SLNs after radioactive colloid is
injected. Moreover, radioactive materials handling is
another challenge for hospital management. A blue dye
may lead to staining of adjacent adipose tissue. In
addition, breast tattooing is another problem that limits
its application. Anaphylaxis following blue dye injection
is also an infrequent but serious risk for patients. In this
study, we chose sulfur hexafluoride microbubbles as a
contrast agent, which is much smaller than red blood
cell; so the agent can readily cross into the SLNs and be
easily eliminated. The detecting instrument is a normal
ultrasound apparatus with contrast pulse sequences, and
it is readily available. Moreover, there are no iodine and
proteins in sulfur hexafluoride microbubbles, which
prevents patients from allergy. In consideration of the
receivable accuracy rate of blue dye guided method [
and its convenience and fast transit, we chose blue dye as
the reference substance for SLNB.
The potential application of CEUS and microbubbles
in identifying SLNs in a swine melanoma model was first
reported in 2004 [
]. Then, a few studies of SLN
identification by CEUS and microbubbles in human beings
were published. A UK study with 54 patients found 89 %
identification rate by CEUS and intradermal
]. This result was repeated in a further
80patient UK study [
]. In 2013, a larger study of 347
patients also found a low sensitivity of 61 % but a
specificity of 100 % [
]. In the above three studies, there was
no significant difference between the identification rates
by CEUS and standard dual technique. However, another
Japan study with 20 patients had a detecting rate of
70 %, much lower than that of γ-probe-guided method
of 100 % [
]. Dual-labeled technique was used as the
control method in all the above studies, but the patient
numbers varied widely and there was also difference in
the effectiveness. In addition, there are few reports on
the relationship between the pattern of enhancement
and lymph node status.
SLNB using blue dye and radioisotope often results in
the removal of multiple SLNs. In this study, the
identification rate of CEUS was 97.03 %. One hundred fifteen
SLNs were excised by CEUS, which was less than blue
dye. The positive rates of SLNs identified by CEUS was
35.7 % (41/115), significantly higher than that by blue
dye (21.8 %, 46/211). Rates of axillary seroma and
infection in patients who had five or more SLNs removed are
significantly higher than those with up to four nodes
]. The characteristic advantage of CEUS and
microbubbles is the real-time imaging for SLNs. A
dynamic process of SLN enhancement from the afferent
lymphatic vessel to efferent vessel is visible, and the very
first developed lymph nodes are easily identified and
located with a guidewire. Therefore, the number of SLNs
detected by CEUS was less than that detected by blue
dye in our study, but there was no significant difference
between the identification rates by CEUS and blue dye.
In other words, maybe SLNB with CEUS can adequately
stage the axilla with fewer traumas. The false negative
rate of SLNB is the most important concern of the
surgeons. It was reported that SLNB had an inherent false
negative rate in the range of 4–10 % [
]. In our
study, all the axillary positive cases were diagnosed
correctly by CEUS except for three undeveloped cases,
which underwent ALND consequently and were proved
axillary positive pathologically. Therefore, no patient
delayed her treatment. There was no unnecessary ALND
in the consequence of CEUS, either. However, one case
was misdiagnosed negative by blue dye, which may lose
the opportunity of ALND without CEUS.
The use of microbubbles and CEUS may also be of
value in the diagnosis of involved SLN. The normal
direction of internal drainage of lymph nodes is from
cortex to cortex, which appeared to be enhanced from
peripheral region to central zone in CEUS. SLNs that
contained US-detectable metastases demonstrated areas
did not enhance as a result of tumor displacement or
destruction of the normal tissue. Goldberg had reported
that the sensitivity and specificity of CEUS in predicting
SLNs metastases in a swine model were 95 and 63 %,
respectively, with an overall accuracy of 86 % [
another 20-case study, inhomogeneous enhancement
was also observed in 2 metastasized cases. Peripherally
increased echogenicity or longer enhancing period was
observed because of the replacement of the inner
portion of the LN by metastatic lesions [
]. However, there
are few reports about the value of the CEUS in
predicting lymph node metastases. In our study, we
summarized the enhancement pattern into three types and
obtained an accuracy rate of 85.2 %, compared with the
pathology result. Although the result is not very
satisfactory, it is successfully correlated with the intra-nodal
filling defects with tumor involved. This may be a valuable
addition to the technique.
Microbubbles and CEUS is a feasible approach for SLN
identification. The enhancing patterns on CEUS may be
helpful to recognize the metastasizing SLNs. This novel
method may be a promising technique for the clinical
application. Larger patient data and multicenter cohort
trials are required and long-term follow-up data on the
safety and therapeutic effect are still needed.
Ethics approval and consent to participate
The study was approved by the institutional ethics
committee of Peking University People’s Hospital (IRB approval
number is 2012045). Informed consent was obtained from
all enrolled patients under the ‘Ethics, consent and
permissions’ heading (The version number is modified revision
Consent for publication
Informed consent for publishing the individual patient
data was obtained from all the participants.
The authors declare that they have no competing interests.
XF collected the data from the patients and performed the procedures from
SLN locating to SLNB. ZDJ analyzed the data. CL designed the study and
performed the SLNB. YLi and YLei performed the CEUS. TFZ and LHJ
performed the SLNB. WShu and WShan provided guidance for the study.
All the authors read and approved the final manuscript.
This work was supported by the Foundation for Scientific Research and
Development of Peking University People’s Hospital (RDC2012-13).
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