In vivo study to determine the efficacy of sentinel node mapping in patients with colon carcinoma using near-infrared laparoscopy after submucosal injection

For more than 100 years, radical lymphadenectomy has been the diagnostic and
therapeutic gold standard for the management of metastatic nodal disease,
despite well-known morbidities such as lymphedema and nerve damage. (Saha et
al. 245S-9S) Whereby lymph node status is the most important factor in the
selection of patients for adjuvant chemotherapy.(Bilchik and Trocha 219-23;Saha
et al. 245S-9S)

Recurrences occur in 20% of the patients without lymph node metastases. (Kelder
et al.;Saha et al. 245S-9S) Most likely due to inadequate lymph node resection
and/or missed nodal metastases on histologic examination.(Bilchik and Trocha
219-23;Saha et al. 245S-9S) With the conventional H&E staining micrometastases
are not detected. They can be detected using IHC or RT-PCR but these
ultrastaging techniques are labour and cost intensive, so it*s highly
impractical to ultrastage all the nodes in a given specimen.
Several studies indicate that micrometastases are correlated with a
significantly poorer prognosis.(Bilchik et al. 568-75;Cote et al.
613-20;Iddings et al. 1386-92;Liefers et al. 223-28) We speculate that although
no recommendation for adjuvant chemotherapy is given for patients with colon
cancer with stage I/II at this time, a subgroup yet to be defined would
probably benefit from adjuvant chemotherapy.

The primary purpose of SLNM in colon cancer is to upstage tumors whose
metastasis would remain undetected by conventional pathological mains and to
identify aberrant lymphatic drainage.

Several studies concerning the feasibility of SLN mapping in colon carcinoma
have been published. The conclusion of the Meta-analysis published by Des Guetz
et al. is that lymphatic mapping appears to be readily applicable. (Des et al.
1304-12) The global sensitivity and specificity of the SLNM were, respectively,
70% and 81%. (Des et al. 1304-12) In the series of CRCs, more than half of the
patients were found to have T3-T4 tumours. With CRCs, it was previously shown
that massive lymph node involvement was the cause of the high false negative
rate of SLNM (Doekhie et al. 854-62). The studies with the highest false
negative rates have also tended to have the greatest proportion of T3-T4
tumours in their study cohorts. Better results in such patients populations is
reported in those studies which specifically excluded those with evident lymph
node disease intraoperatively (Tuech et al. 158-61) In our study we will
stratify patients according to their T stage. Before routinely using this
technique in CRC it has to be improved. The dyes used in these studies are
patent blue/ isosulfan blue, radioisotope tracers or a combination of one of
these three. In our study we have the possibility to use several tracers such
as; blue dye (patent blue), radioactive (human serum albumin, technetium) or
fluorescent tracers (ICG). Patent blue and radioactive colloid is mostly used
in European studies. So they are well known and safe. ICG is also well known in
the use for other purposes. It has one big advantage. ICG has a peak spectral
absorption at 800-810 nm in blood plasma and blood. Wavelengths in the 800 nm
range penetrate relatively deeply into living tissue compared to visible light,
so SLN staining can also be accurately assessed in mesenteric adipose tissue.
The particle size of ICG is 7,3 nm. The lymphatic collection process is
strongly related to particle size. Particles smaller than 4-5 nm will penetrate
capillary membranes and therefore may not migrate through the lymphatic
channel. Particles larger than 4-100 nm show the most rapid dispersal from
interstitial space into lymphatic vessels, yet have significant retention in
the lymph node. Particles larger than 500nm have a much slower rate of
clearance from the interstitial space with significantly less accumulation in
lymph nodes. The 40 nm beads were considered to be the most appropriate size
for SN detection in rats (Nakajima et al. 353-56). To obtain the optimal
particle size we might make a conjugate with Nanocoll ® (95% of the particles <
80 nm).
For the visualisation of indocyanine green we will have the premiere of using
the first near-infrared laparoscope in Europe (Olympus Corp., Tokyo, Japan).
This scope is being designed specific for this study. It consist of a regular
scope with a special light source and light filters.

Hypoteses:
1) Intra-operative lymphatic mapping and sentinel lymphadenectomy is feasible
in colon cancer.
2) the sentinel lymph node (SLN) status reflects the regional node status
3) focused analysis of the SLN improves staging accuracy
4) micrometastasis have prognostic importance

Primary objective:
To study the faesibility of SLNM in patients with histological proven colon
cancer using NIR-laparoscopy.


Secondary objectives:
To study the incidence of micrometastasis with the use of ICG and RT-PCR
To determine the number of false-negative SLNs
To evaluate the efficiency of minimal invasive surgery for harvesting sentinel
lymph nodes.
To determine the accuracy of the technique

cross-sectional study

Colonoscopy will be performed at the operating room when patients are under
general anesthesia. When the tumor is localized, their will be one injection
with the fluorescent tracer at the base of the tumor. Prior to the
administration n of the fluorescent tracer, there will be an injection with a
small amount of saline to guarantee correct placement of the needle. After
injection of the fluorescent tracer there wil be flushed with a small amount of
saline. Afterwards laparoscopic access will be obtained in the traditional
fashion and abdominal exploration shall be performed to rule out
intra-abdominal metastasis. The involved segment is mobilized carefully without
disruption of the lymphatic channels and blood vessels. The segment will be
inspected for fluorescent nodes with the Near-Infrared laparoscope. These nodes
will be marked with a suture. After mobilization is completed the involved
segment of the colon and the regional lymph nodes will be resected through a
minilaparotomy like the conventional method.
Ex vivo the specimen will be searched for fluorescent spots by the NIR camera
system for fluorescent nodes missed in vivo. Ex vivo identified fluorescent
nodes will be marked with a suture in another colour than sutures used in vivo.
Thereafter, the specimen is opened at the anti-mesenteric side. After the
tumour is identified 0.5 - 2 ml of Patent Blue V dye is injected into the
submucosa circumferential to the tumour, using a tuberculin syringe. The
injection sites are then gently massaged for five minutes to push the tracer
into the lymphatic vessels. Blue coloured sentinel nodes are identified and
marked with a suture in a different colour then the in vivo and ex vivo used
sutures.
Hereafter the whole specimen will be placed in formalin for 24-48 hours.

The treatment of the malignancy will be performed conform the conventional
method.
Yet prospective studies are needed to evaluate the potential benefit of
systematic chemotherapy in patients with micrometastases. At this moment we are
evaluating if it*s favorable to randomize patients with micrometastases for
adjuvant chemo.