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ORIGINAL ARTICLE
Near infrared fluorescent imaging after intravenous injection of indocyanine green
during neck dissection in patients with head and neck cancer: A feasibility study
Antoine Digonnet, MD,1* Sophie van Kerckhove, MSc,2 Michel Moreau, MD,3 Esther Willemse, MD,1 Marie Quiriny, MD,1 Bissan Ahmed, MD, PhD,2
Nicolas de Saint Aubain, MD,4 Guy Andry, MD,1 Pierre Bourgeois, MD, PhD2
1
Department of Head and Neck Surgery, Jules Bordet Institute, Universite Libre de Bruxelles, Brussels, Belgium, 2Department of Nuclear Medicine, Jules Bordet Institute, Universite Libre de Bruxelles, Brussels, Belgium, 3Department of Biostatistics, Jules Bordet Institute, Universite Libre de Bruxelles, Brussels, Belgium, 4Department of Pathology,
Jules Bordet Institute, Universite Libre de Bruxelles, Belgium.
Accepted 20 September 2015
Published online 24 December 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.24331
ABSTRACT: Background. Indocyanine green (ICG) has not been studied
during therapeutic lymph node dissections after intravenous injection.
The purpose of this study was to explore the distribution of ICG in lymphatic nodes during neck dissection.
Methods. Eleven patients requiring neck dissection with or without
resection of the primary lesion were included. ICG was intravenously
injected at induction time of anesthesia. Imaging was performed before
and after surgical resection. Fluorescence was measured in arbitrary
units (AUs) in the pathology department. Mixed linear model and generalized estimating equations (GEEs) were used.
Results. Mean fluorescence of invaded nodes was 22.6 AUs (SD 5 24.9)
and 3.9 AUs (SD 5 8.1) in negative nodes (p 5 .016). After adjustment
for the size of the node, the risk of invasion when fluorescence
was observed was 12.2 (95% confidence interval [CI] 5 5.3–28.2;
p < .0001).
Conclusion. This study demonstrates the feasibility of ICG to bring a contrast during surgery between healthy and invaded nodes after i.v. injecC 2015 Wiley Periodicals, Inc. Head Neck 38: E1833–E1837, 2016
tion. V
INTRODUCTION
To the best of our knowledge, NIR has not yet been studied in therapeutic lymph nodes dissection after intravenous (i.v.) injection. The purpose of this study was to
evaluate the feasibility and the distribution of ICG in the
lymph nodes after i.v. administration.
The nodal status in head and neck squamous cell carcinoma remains one of the most important prognostic factors.1,2 Proper treatment of the neck compartment is a
critical issue because leaving behind invaded nodes could
lead to earlier recurrence and death. The gold standard
for lymph node disease is neck dissection.3 The use of
near infrared (NIR) fluorescence light has been recently
used to intraoperatively identify lymph nodes, tumors,
and vital structures.4 NIR fluorescence using the fluorescent dye indocyanine green (ICG) has been successfully
used for sentinel lymph node mapping in head and neck
squamous cell carcinoma.5,6 Unfortunately, regarding
tumor location, sentinel lymph node mapping is not
always technically possible in head and neck cancer.
Peripheral vein injection of ICG has shown to be effective to identify macroscopically invaded nodes and primary tumor.7 The optimal timing for carrying out the
surgical procedure is between 30 minutes and 2 hours
after i.v. ICG administration in head and neck cancer.7
*Corresponding author: A. Digonnet, Department of Head and Neck Surgery,
Jules Bordet Institute, Universite Libre de Bruxelles, 1 rue Heger Bordet, 1000
Brussels, Belgium. E-mail: [email protected]
Contract grant sponsor: This work was supported by “Les amis de l’institut
Bordet” and group Research and Development; “Clinical Applications Fluorescent Imaging” (coordinator: P.B.)
KEY WORDS: indocyanine green, lymph node, neck dissection,
oncology, fluorescence abstract
MATERIALS AND METHODS
This clinical trial was approved by the medical ethic
committee of the Institute Jules Bordet, Universite Libre de
Bruxelles. Patients with a history of renal failure and coronaropathy were excluded. Eleven patients from a single
institution were prospectively enrolled. Decision of the
neck dissection with or without a resection of the primary
tumor was taken during our multidisciplinary oncologic
consultation. Free ICG (0.25 mg/kg; Pulsion Medical System, Belgium) was injected through the radial vein at
induction time of anesthesia. The setting in the operating
room was performed according to the procedure described
by Mieog et al8 and we used a dedicated NIR camera
device (PDE, Hamamatsu, Japan). Patients requiring resection of a primary lesion underwent fluorescent tumor
inspection before and after resection. After subplatysmal
flap elevation, the neck operative field was inspected with
the NIR camera to identify potential hotspots, and then the
procedure was carried out as previously planned. At the
end of the neck dissection, the operative field was
re-inspected with the infrared camera, and potential
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TABLE 1. Patient characteristics.
Patient
number
Age,
y
Primary tumor
Preoperative
TNM classification
Resection of the
primary
1
2
3
49
63
68
Larynx (glottis)
Oropharynx
Larynx-esophagus
T4N0M0
T2N2b
T1N0 and T2NO
55
68
53
60
66
63
44
45
Larynx
Piriform sinus
Lip
Tongue
Thyroid
Thyroid
Melanoma
Melanoma
T2N0
T4aN3
T2N0
N1
N1a
T3N1b
IIIb (N1b)
IIIb (N1b)
Partial laryngectomy
Pharyngectomy
Eso-pharyngolaryngectomy
Total laryngectomy
No
Resection
No
No
Total thyroidectomy
No
No
4
5
6
7
8
9
10
11
hotspots in the resected area were harvested. The hotspot
identified outside the planned resected area was also
harvested.
The resected specimen was sent for macroscopic analysis, the number of nodes were counted, and the surface of
the nodes were measured (in mm2) according to their
great and small diameter, and then the specimen was
reexamined under fluorescence and the nodes were recounted. Fluorescence of each resected node was measured in arbitrary units (AUs). Finally, a microscopic analysis was performed for each node to determine its status
(invaded or not).
Statistical analysis
Fluorescence of the nodes was analyzed as a continuous
and categorized variable using cutoffs of 6 UAs (6/<6).
As the size of the node was not the variable of interest
but only an adjustment variable, the analysis was performed in a continuous way.
As the unit of analysis was the lymph node and not the
patient, and the data are not independent, we used the
mixed linear model to test our hypothesis (for continuous
variable) and generalized estimating equation (GEE)
model (for categorical variable) in order to take into
account the correlation structure within patient. Odds
ratios (ORs) were derived with their 95% confidence
intervals (95% CIs). In the mixed linear model, compound symmetry was used as the type of covariance
matrix. In order to have a symmetrical distribution of
dependent variables, we used the log-transformation of
the fluorescence and the size.
In the GEE analyses, empirical instead of model-based
SEs were used because they are more robust against misspecification of the correlation structure. The exchangeable covariance matrix was used.
Fluorescence was analyzed in univariate and multivariate (adjustment for the node size and histology).
RESULTS
Patients and tumor characteristics
This study was proposed to 13 consecutive patients.
Two patients refused the study and 11 patients were
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Size of the
resected
primary, cm
Neck dissection
No. of
harvested
nodes
No. of
invaded
nodes
1.8
3.5
1.3
II–III (bilateral) VI
II–III
VI
28
8
4
0
2
0
2
II–III–Va (bilateral)
V
I
II–III–Va
I retropharyngeal
II–III–IV–V
III–IV–Vb
III–IV–Vb
19
6
2
21
6
57
17
25
0
1
0
1
1
9
2
1
2.2
6
included and injected. The mean age was 58.9 years
(range, 44–68 years). Patients, tumors characteristics, and
the performed procedure are detailed in Table 1.
Six patients underwent resection of the primary lesion
with a neck dissection during the same procedure. Among
them, 2 patients underwent salvage surgery. Patient number 3 was previously treated by exclusive radiotherapy for
glottis cancer and 7 years later he underwent a partial laryngectomy with neck dissection for recurrence. During
follow-up, an esophageal cancer was diagnosed and
treated by chemoradiotherapy (CRT). One year after
CRT, the patient experienced recurrences on both sites
leading to a total esophageal laryngectomy. Patient number 4 was previously treated by radiotherapy for epiglottic
lesion; he experienced a first recurrence treated by transoral robotic surgery (epiglotectomy). The last recurrence
was again supraglottic and treated by total laryngectomy.
The 5 remaining patients were operated on for lymph
nodes metastasis.
Patient number 5 had a T4aN3 of the piriform sinus.
He refused total laryngectomy and was enrolled to receive
induction chemotherapy. After 2 cycles of docetaxel, cisplatin, and 5-fluorouracil, he experienced a persistence of
nodes in level V. He underwent level V dissection and
was sent for concurrent CRT.
No adverse reactions or complications related to the
ICG injection occurred during the current study.
Timing of fluorescence
Table 2 details the timing (Delta) between ICG and the
initial inspection with the fluorescence camera. Two
patients (8-9) underwent initial inspection after 2 hours
and were those who required 2 separate incisions to perform the planned neck dissections. Using GEE analysis,
fluorescence (AU) was not correlated to the Delta
(minutes; p 5 .64).
Perioperative imaging
Before neck dissection, the lymph node compartment
was subjectively fluorescent in all patients with the
exception of 1 patient (patient 3). An example of intraoperative fluorescence is shown in Figure 1. Fluorescent
examination of the resected area showed no residual
INDOCYANINE
TABLE 2. Timing between injection time and initial inspection.
Patient number
1
2
3
4
5
6
7
8
9
10
11
Injection time
9 h 38
12 h 35
14 h 22
9 h 14
11 h 17
14 h 13
12 h 00
9 h 10
11 h 30
10 h 31
11 h 48
Initial inspection
11 h 22
13 h 59
16 h 02
11 h 05
12 h 05
14 h 45
12 h 30
10 h 56–13 h 28
13 h 45
11 h 30
13 h 05
GREEN IN THERAPEUTIC LYMPH NODE DISSECTION
TABLE 3. Sensitivity and specificity.
Delta, min
104
84
100
111
48
32
30
106–258
165
59
77
fluorescence, thus, no additional lymph node was harvested in this field. One patient had a hotspot outside the
planned resection area and 1 had 2 hotspots. Additional
resection revealed 1 benign lymph node, 1 fatty tissue,
and a muscle fragment.
Regarding primary tumor examination, thyroidectomies
were excluded because margin evaluation was less relevant. Primary tumor examination (n 5 5) revealed fluorescence in all patients with the exception of 1 patient
(patient 3). Fluorescent imaging after resection of the primary lesion showed residual fluorescence in 1 patient
(patient 6) and frozen section confirmed margin involvement leading to complementary resection. Margins were
Node status
Positive
Negative
13
4
17
41
135
ICG imaging
Positive
Negative
54
139
Abbreviation: ICG, indocyanine green.
Sensitivity: 13 of 17 5 76.5%.
Specificity: 135 of 176 5 76.7%.
clear after the second resection and no residual fluorescence was found.
The remaining 3 patients without residual fluorescence
after resection of the primary tumor had immediately
clear margins.
Histopathologic analysis
Fluorescence imaging did not modify the lymph node
count in the department of pathology.
Lymph nodes
Across the 11 patients, 193 lymph nodes were harvested with an average number of 17.5 lymph nodes (SD
5 16.1) per patient and a median of 13. Five patients
(45.4%) had <10 resected nodes, 2 (18.2%) had between
10 and 20 resected nodes, and 4 (36.4%) had >20
resected nodes.
Among the 193 lymph nodes, 17 were positive (8.8%),
54 (28%) were fluorescent (AUs 6). The mean fluorescence and size of the 193 lymph nodes were respectively
5.7 UAs (SD 5 12.1) and 94.6 mm2 (SD 5 126.5).
In invaded lymph nodes, the mean fluorescence was
22.6 UAs (SD 5 24.9) and 3.9 UAs (SD 5 8.1) in negative lymph nodes (p 5 .016). For the size, we had 238.2
mm2 (SD 5 244.6) and 78.6 mm2 (SD 5 94.5), respectively, with p 5 .011. The median fluorescence in positive lymph nodes was 11 AUs (range, 0–86 AUs). The
median size of metastatic deposits that were ICG-positive
was 120 mm2 (range, 20–900 mm2). We observed a correlation between amount of ICG uptake and the size of
the metastatic nodes (p 5 .02). Sensitivity and specificity
of the test with ICG were 76.5% (13 of 17) and 76.7%
(135 of 176), respectively (Table 3).
In univariate analysis (GEE), a fluorescence of 6 AUs
or more is associated with a 10.3-fold (95% CI 5 4.8–
22.1) increased risk to have a positive node (Table 4).
After adjustment for the size of the lymph node, the
OR for invasion in case of fluorescence was 14.1 (95%
CI 5 4.6–42.8).
Preoperative workup, and fluorescent and invaded
lymph nodes
FIGURE 1. Perioperative view using near infrared (NIR) imaging
for patients 2 and 10 improved the quality of the image compared
to patient 2 assessing the existence of a learning curve (a fluorescent node is seen between the grasping forceps).
Table 5 details the number of suspicious nodes found on
preoperative staging, the number of invaded nodes that were
ICG fluorescent, and the total amount of invaded nodes.
Table 5 demonstrates that there were 3 invaded nodes
detected by ICG but were not detected at preoperative
workup (patient 9). In the same patients, there were 4
invaded nodes that were not ICG fluorescent.
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TABLE 4. Univariate analysis (generalized estimating equation).
Variables
Fluorescence 6
Positive
node
number (%)
OR
Lower
95% CI
Upper
95% CI
p value
13 (7.3)
10.3
4.8
22.1
< .0001
Abbreviations: OR, odds ratio; 95% CI, 95% confidence interval.
In patient 11, we also saw that we had a false-positive
result on positron emission tomography-CT, this was not
the case with MRI, ultrasonography, or ICG.
DISCUSSION
The use of ICG in human oncology started in 2000 in
breast cancer.9 Further studies demonstrate the ability of
ICG to bind tumor tissues.10,11 ICG was first described in
head and neck oncology after peritumoral injection for
sentinel lymph node mapping.5,6 Anatomic constraints of
head and neck cancer localization, particularly below the
oropharynx, complicated the routine use of this procedure.
In 2013, Yokoyama et al7 demonstrated the property of
ICG to bind head and neck tumoral tissue after i.v. injection. The optimal timing for surgery was found to be
between 30 minutes and 2 hours after i.v. injection. In
line with this result, we decided to perform the injection
at the induction time of the anesthesia. In our series, 2
patients underwent the initial fluorescence inspection after
2 hours; however, we did not find a decreased fluorescence in those patients compared with the rest of the population. The optimal timing, set by Yokohama et al,7 used
subjective scales of fluorescence, which may have led to
a decrease in precision.
Regarding the administered preparation, some authors
have mixed ICG with human serum albumin to obtain
better retention in the lymph nodes.12 We decided to
inject free ICG because there is no argument for the superiority of mixed ICG to serum albumin compared to free
ICG.13
The principal finding was the correlation between node
involvement and fluorescence magnitude. In their study,
Yokoyama et al7 demonstrated the ability of NIR imaging
to distinguish cancer from normal surrounding tissue.
However, they only dealt with macroscopic lesion identifiable by sight or palpation. In the present study, we
found that fluorescence was associated with a 14.1-fold
risk of invasion regardless of the size of the node. In
summary, fluorescence was correlated to invasion status
and, to a very small extent, to the size of the node.
According to our knowledge, this is the first study to
bring out a perioperative contrast between invaded and
healthy nodes after i.v. injection of ICG.
The second finding was the observation of an ICG distribution in healthy nodes resulting in subjective fluorescence. This result allows delimitation between the nodes
and the surrounding tissues. However, in this preliminary
study, NIR imaging did not increase lymph node yield
during neck dissection because visualization of the resection site demonstrated no residual fluorescence. Surprisingly, we observed discordance between subjective
fluorescence and objective measurement of the healthy
nodes’ AU value (mean, 3.9 UAs). This result may be
explained by the fact that fluorescence was measured in
the department of pathology. This condition has probably
led to a decrease in the measure of fluorescence.
Because tissue layers are thin in the neck compartment,
NIR imaging may also help to identify residual invaded
nodes outside the resected area once the neck dissection
is completed. However, per-operative fluorescence
remains subjective because AUs are measured postoperatively. If the device imaging seemed to be simple to handle and a few were time-consuming for the surgical
procedure, we believe that a learning curve is associated
with the perioperative setting of the device and interpretation of fluorescence (Figure 1). In the future, we could
elaborate a subjective scale of fluorescence and compare
it to objective UA measurements.
The third finding was the potential relationship between
fluorescence and margin status. Indeed, in our series, 5
patients underwent resection for primary head and neck
squamous cell carcinoma. Four patients had fluorescent
tumors and imaging of the resected field revealed residual
fluorescence in 1 patient. Correlation with frozen section
biopsy showed only invasion in the patient with residual
TABLE 5. Number of suspicious nodes found on preoperative staging, number of invaded nodes indocyanine green fluorescent, and the total amount of
invaded nodes.
Patient
number
1
2
3
4
5
6
7
8
9
10
11
No. of suspicious nodes (imaging)
CT
MRI
PET-CT
0
2
0
0
1
0
Ultrasonography
2
0
0
1
3
1
2
1
2
2
2
1
2 1 FNA
1 1 FNA
Abbreviations: PET, positron emission tomography; ICG, indocyanine green; FNA, fine-needle aspiration.
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No. of suspicious
nodes (preoperative)
No. of invaded
nodes ICG1
No. of invaded
nodes
0
2
0
0
1
0
1
3
2
2
2
0
2
0
0
1
0
1
1
5
2
1
0
2
0
0
1
0
1
1
9
2
1
INDOCYANINE
fluorescence. The 3 remaining patients had healthy margins at frozen section examination. This finding, if confirmed by a larger series, could establish that NIR
fluorescence imaging has the potential to help improve
tumor resection compared with current intraoperative
methods. In a recent study, Martirosyan et al14 studied
animal models of glioblastoma. They performed tumor
resection with the use of NIR laser confocal endomicroscopy with ICG. They found that ICG provides striking
distinguishing features of normal brain and tumor regions,
providing definitive tumor boarder delimitation.
In our series, 1 patient (patient 3) did not experience
node and primary tumor fluorescence after i.v. injection
of ICG. This patient underwent 2 sessions of radiotherapy
and 1 surgery with node dissection before our last procedure. We can assume that those previous treatments might
have impaired the biological property of tissue and lymphatic vessels leading to a decreased fluorescence.
Imaging of the operative field after neck dissection did
not reveal residual nodes in the resected area, but hotspots
were found outside of those limits. Resection of those
hotspots provides poor information because we found 1
node, 1 fatty fragment, and 1 muscle piece. These results
could be interpreted by a nonspecific tissue fixation
increasing with time because imaging was performed at
the end of the procedure.
In conclusion, NIR imaging was easily achievable without disturbing the surgical procedure. NIR imaging after
i.v. injection of ICG offers the ability to visualize the
neck node compartments during surgery. Fluorescence
magnitude was correlated to node invasion and brings a
contrast between healthy and invading nodes after i.v.
injection.
Regarding the primary lesion, NIR imaging could help
to define the tumor border and guide the surgical
resection.
GREEN IN THERAPEUTIC LYMPH NODE DISSECTION
Larger and homogenous series are required to define
the optimal role of NIR fluorescence in head and neck
cancer and its potential routine utilization.
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