Sentinel lymph node identification in colon cancer using the hybrid tracer ICG- 99mTc-nanocoll

Colon cancer is one of the leading causes of cancer related deaths in the
Western World. In the Netherlands there are over 10.000 new cases of colon
cancer each year which will increase to more than 14.000.
Due to the introduction of nationwide screening programs the incidence of stage
I and II disease is expected to increase. Local excision of the primary tumor
is an attractive treatment option but is only oncological safe in the absence
of lymph node metastasis. The current standard preoperative imaging modalities
(CT and MRI) are not able to distinguish nodal involvement. As a result all
patients need to be treated with a resection of the primary tumor and
appropriate en-bloc resection of associated lymph nodes. Fifty precent of the
diagnosed patients suffers from a stage I/II disease. The extensive surgical
approach leads to over-treatment and unnecessary exposure of surgery-related
morbidity in especially these patients with early CRC. However, the 5-year
survival rates for patients with stage I and II colon carcinoma are
respectively 90% and 75%. Unfortunately, up to 30% stage I/II patients will
nonetheless develop distant metastases and eventually die from colorectal
carcinoma. This is probably caused by understaging of the LNs since occult
tumor cells and micrometastasis, associated with disease recurrence and poor
survival, but easily missed during routine hisptopathological examination
(single-section haematoxylin and eosin staining). Ideally, all lymph nodes
should be examined with serial sectioning and additional immunohistochemistry
or reverse transcriptase RT-PCR for more accurately staging. Unfortunately,
these techniques are expensive and time-consuming which make them not
preferable in daily practice. Apart from insufficient pathologic examination of
the LNs, lymphatic drainage patterns are variable or can be aberrant from
conventional resection margins which lead to incomplete lymphadenectomy.
The aim of this project is to design an easy and widely applicable method to
improve lymph node metastases detection techniques.

Drawbacks of current lymph node staging
Better detection and pathologic staging of the lymph nodes will contribute
significantly to a better survival of colon cancer patients. There are a number
of drawbacks in the current lymph node staging technique which makes it
difficult to classify and stage patients accurately and withhold the optimal
postoperative treatment.
First, on standard preoperative imaging it is often difficult to determine if
lymph nodes contain metastases. During surgery, the lymph nodes are too small
(± 0.5 mm diameter) and therefore not visible to the naked eye. Moreover, the
pathologist identifies the lymph nodes by palpation. Because of the very small
size of the lymph nodes from CRC, they are easily missed. Second, understaging
of the lymph nodes by histopathological examination may also contribute to
inadequate staging. Usually, the lymph nodes are cut in two parts and only one
slice from both halves is examined.
(Micro-)metastases outside these slices are missed. Ideally, all lymph nodes
should be examined on different levels by cutting the lymph nodes in several
parts. Although staging can be improved by addition of advanced pathological
staging techniques like immunohistochemistry or RT-PCR, these additional
techniques are too expensive and time consuming to analyze all lymph nodes.
This is therefore not feasible in every day practice.
The sentinel lymph node (SLN) concept could offer a solution. This procedure
aims to identify the first 1-4 lymph nodes that have the most direct drainage
from the primary tumor and therefore contain the highest risk of harboring
metastases even when located outside the resection area. Identification of the
SLN allows the pathologist to examine these few nodes with additional
histopathological techniques as mentioned above.
Sentinel lymph node procedure in colon cancer
The SLN procedure is a diagnostic staging procedure in multiple types of cancer
like melanoma and breast cancer. A SLN with metastatic disease is an indication
for additional extensive surgery or adjuvant postoperative chemotherapy.
However, a negative SLN would justify a wait and see policy. Treatment of
breast cancer and melanoma patients with the SLN procedures has proven to be a
safe and valuable technique. Since the introduction of the SLN procedure, it
changed the treatment of these cancers and improved the quality of life of
these patients dramatically.
Many studies already support its feasibility but the sensitivity rates are low
and variable. Van der Pas et al. (1) showed in meta-analysis that after
selection of high-quality studies, an increase of sensitivity up to 90% is
possible when the SLN technique would be standardized and patients selection
refined.
First, colon and rectal cancer should be separated since the majority of
patients with rectal cancer receive neoadjuvant therapy which might obliterate
lymphatic flow. Secondly there are some technical difficulties which counteract
clinical implementation of SLN mapping in CRC. The current most used optical
dyes are patent blue or isolfan blue which have been used in an in vivo and ex
vivo settings. Both blue dyes cannot be seen through fatty tissue and easily
diffuse trough the true SLN and stain regional lymph nodes. Also the injection
technique varies between studies.
Using radioactive tracers presents the problem of signal interference of the
injection site. The very strong signal of the injection site *overshines* the
signal of the SLNs which are located near the tumor which makes SLN detection
impossible. Besides that, the limitation to visualize the lymph nodes and his
corresponding lymphatic vessels during surgery itself makes radioactive tracers
alone less attractive.
Ideally the SLN procedure should be performed in vivo to overcome the problem
of aberrant lymphatic drainage. Subserosal injection of dye can be easily
performed in vivo but tumor margins are not very clear during this approach.
Subsequently, dye injection occurs easily to far from the tumor or into the
site which do not exactly match the lymphatic drainage from the malignancy. An
endoscopic submucosal injection allows for better tumor visualization which
results in more accurate dye administration and lymphatic drainage pattern
towards the SLN. Although an additional pre- or perioperative colonoscopy is
necessary this disturbs and prolongs the surgical procedure.
SLN detection with a near-infrared (NIR)-imaging and the fluorescent dye
Indocyanine Green (ICG) is a new and promising technique which may have the
characteristic to solve all the problems inherent to use of blue dye and
radioactive tracers. NIR-light penetrates deeply into fat tissue compared with
visible light or blue dye. It can be used during the surgical procedure and can
be seen during the operation. This provides the opportunity for the surgeon to
localize the lymph node in the abdomen and makes sure there is an adequate
resection.
Preliminary results
Our research group investigates the applicability of the SLN procedure in
colorectal cancer since 2011. To identify the SLN we use the Near-Infrared
(NIR) dye Indocyanin Green (ICG). Great advantage of the NIR-dye compared to
conventional light or blue dye is the better penetration depth which makes
deeper located structures visible.
To investigate the feasibility of SLN detection with the NIR technique, we
started in a goat model (2). In this study we identified SLNs after injection
with the fluorescent dye Indocyanin Green dissolved in saline (0.9%) and human
albumin (200 mg/ml) with a new developed laparoscopic NIR fluorescence imaging
system. Hereafter we performed a feasibility study to establish the SLN
technique in colon cancer patients with no signs of (lymph node) metastases. We
compared two injection techniques. In fourteen

Aim of the study is to develop an intraoperative technique for SLN mapping in
colon cancer which is suitably in daily practice in colon surgery in an average
hospital setting.

The study will be a prospective non-randomized trial in which we use the hybrid
tracer 99mTc-Nanocoll-ICG to detect the SLN in patient with colon cancer. The
study will focus on upstaging and applicability in daily practice.
When this SLN technique is accurate and feasible if will be used in a large
randomized clinical trial.

Patients will be injected with the tracer 99mTc-Nanocoll-ICG respectively 5 or
14 hrs before surgery. The tracer will be administrated by colonoscopy
performed by the gastroenterologist and injected at the base of the tumor in
submucosal layer. After tracer administration, patients undergo a SPECT-CT scan
just before the surgical procedure. The SLN procedure will be performed during
the conventional resection in a adjusted sequence. First the segment will be
inspected for fluorescent nodes with the Near-Infrared- laparoscope (Olympus
Corporation, Tokyo, Japan). Fluorescent nodes are marked with a suture.
Secondly, the segment will be inspected with the gamma-probe. Radioactive nodes
are marked with a suture too. Thereafter the number of fluorescent and/or
radioactive nodes found in vivo will be compared with the number of nodes found
by preoperative imaging. After removal the specimen, it will be inspected for
fluorescent and/or radioactive nodes which are not detected inside the body.
These nodes will also be marked with a suture. To confirm if the intraopertive
marked SLN are the same as seen on preoperative SPECT/CT, an additional
SPECT/CT of the specimen will be performed. The entire specimen will be
submitted for pathologic examination. The SLNs will be taken out first and
fluorescence and radioactivity will be confirmed by using the NIR-laparoscope
and gamma-probe. All non-marked regional lymph nodes found will also examined
for fluorescence and radioactivity. All identified SLNs and regional lymph
nodes will be stained with hematoxylin-eosin (H&E). If the sentinel lymph nodes
are negative after routine H&E staining, they will be sliced in multiple parts
and examined with H&E staining and immunohistochemistry with the specific
marker CAM5.2 and CK19.
Metastases between 0.2 mm and 2.0 mm will be referred to as micrometastases
(N1mi). Metastases smaller than 0,2 mm will be described as isolated tumor
cells (N0i+). Lymph nodes which are not fluorescent or radioactive will be
screened according to the SLN protocol too.
Patients will be considered as N+ when metastases are found after multislicing
& staining. These patients are eligible for adjuvant chemotherapy. There are no
treatment implications for patients in which we found micrometastases or
isolated tumor cells. Follow-up of patients will be five years.
Ten patients will be included.

Additional risks for the patients are negligible. The overall risk of colon
perforation due to the colonoscopy is
<0.002%. The effective dose for patients will be 1.25 mSv in total; 0.5 mSv
from 100 MBq 99mTc-nanocoll-ICG and 1.5 mSv from the low-dose SPECT-CT
scans. The effective dose for the gastroenterologist will be 2 uSv/hr when a
distance of 50 cm from the patients will be maintained. The mean duration of a
colposcopy is 30 minutes. The effective dose for the surgeon will be less than
0,1 mSv/hr for each patient. Other medical professionals who contact the
patient will not receive more than 0.008 mSv during the treatment of one
patient for each patient. The maximum allowed effective dose for these persons
is 1 mSv/year. We will not exceed this amount. All medical professionals
involved in the study will not exceed the maximum allowed radiation of 1000
uSv/year.