A pilot study to assess the safety and feasibility of fluorescent sentinel lymph node identification in colon carcinoma using submucosal bevacizumab-800CW

The current gold standard for the treatment of colon carcinoma consists of the
surgical en-bloc resection of the colonic segment including the adjacent
mesocolon containing the draining lymph nodes. Analysis of these lymph nodes is
important, since lymph node status is one of the most important prognostic
factors determining the use of adjuvant chemotherapy. Although patients with
tumour stage I and II do not have lymph node metastases, 15-20% develop
recurrent disease. Several studies suggest that ultrastaging techniques such as
immunohistochemistry (IHC) or reverse transcriptase polymerase chain reaction
(RT-PCR) using multilevel slicing results in upstaging of 14-18% of patients,
due to newly found (micro)metastasis. Furthermore, several studies indicate
that these micrometastases are correlated with a significantly poorer
prognosis, subsequently suggesting that this subgroup of patients might benefit
of adjuvant chemotherapy. Therefore, the most recent Dutch guidelines advice
the use of adjuvant chemotherapy in this *upstaged* group, although evidence is
still lacking.
However, ultrastaging techniques are labour-intensive and costly, and therefore
not suitable for analyses of all lymph nodes that have been collected during
segmental colectomy. Sentinel lymph node (SLN) identification in colon
carcinoma has been proposed to overcome this problem by identifying the first
order draining lymph node(s) of the tumour, which have the highest chance of
containing metastatic tumour cells. Several studies aimed at SLN identification
in colon carcinoma have been published, however, early studies using
radio-guided or blue-dye guided SLN identification, showed relatively high
rates of false negatives with consequent low sensitivity rates. Since mesocolon
is rather fatty tissue, visualization of conventional dyes is difficult.
Indocyanine green (ICG), which can be visualized using near infrared (NIR), has
been put forward since it is known to penetrate relatively deep into living
tissue.
Nevertheless, results of SLN identification using ICG remain unsatisfying with
high false-negative rates and low sensitivity. Most likely this is due to the
fact that these studies also included large cT3-cT4 tumours and patients with
massive lymph node involvement. Which are factors known to interfere with lymph
drainage patterns. Furthermore, subserosal injections were frequently used,
while it is suggested that submucosal injections might result in better
sensitivity of the procedure. In the FLUOR-SLN-ICG pilot study, we successfully
conducted SLN identification in patients with ICG. We want to expand the pilot
study using a tumour-targeted tracer: bevacizumab-800CW. Bevacizumab-800CW can
be preoperatively administered, binds to tumour and metastases, thus allowing
more time for uptake in patients with larger tumour and lymph node metastases.
Therefore this prospective study aims to assess the safety and feasibility of
lymph node identification using bevacizumab-800CW in patients with cT1-3N0-2
tumours, using peritumoral submucosal injections.

The primary outcome parameters are identification rate of SLN(s) or lymph node
metastases with bevacizumab-800CW, defined as the number of patients in which a
SLN or lymph node metastasis was detected due to fluorescence during surgery
and/or pathology assessment divided by the total number of procedures.
Furthermore the rate of adverse events related towards bevacizumab-800CW will
be measured. This is defined as the number of adverse events related towards
bevacizumab-800CW/total number of procedures.
Secondary outcome parameters include: amount of fluorescence in lymph node
metastases compared to lymph node without metastases, false-negative SLNs,
true-negative SLNs, sensitivity, upstaged patients, aberrant lymph node status,
accuracy, negative predictive value and number of SLNs identified.

This is a single-centre, open-label, non-randomized cohort safety and
feasibility study.

1. Patients are identified at the outpatient clinic and asked for participation
in the study.
2. Patients will be planned for laparoscopic/robot-assisted surgical colectomy
according to standard of care (SOC).
3. 2-4 days before surgery, a colonoscopy is performed by the
gastroenterologist to submucosally inject a maximum of 4.5mg bevacizumab-800CW
around the tumour. The patient is observed during one hour after injection of
bevacizumab-800CW. Before the colonoscopy, mechanical bowel preparation (MBP)
will take place according to hospital protocol.
4. During segmental colectomy, a NIR camera is used to visualize the SLN,
which will be marked using a stitch. If an aberrant lymph node is visualized,
this node will be harvested.
5. Segmental colectomy with procurement of the adhering mesocolon will be
performed according SOC.
6. After extraction of the specimen, ex-vivo examination of the specimen using
the NIR camera will be performed.
7. Postoperative management will be according SOC.
8. Pathological examination will be done using haematoxylin & eosin (H&E). If
no lymph node metastases are found, the lymph nodes will be examined using
serial slicing and subsequent IHC.
9. Resected specimens or formalin-fixed paraffin embedded blocks of tissue will
also be examined in the UMC Groningen for the presence of NIR. This process
will not interfere with standard working procedures related to clinical care.
In case no SLN can be detected with intraoperative fluorescence, we will be
able to detect (ex vivo) fluorescence with specially designed cameras present
at the UMCG. These cameras can detect bevacizumab-800CW while more than
>16.000x diluted. This allows for *ex-vivo* SLN identification if there is any
clinically relevant fluorescence present in the lymph nodes, while no
intraoperative fluorescence is detected. Therefore, these results then still
can be translated to the clinic when optimised systems become available.

This study is a non-therapeutic, diagnostic feasibility study, aimed at
identification of the SLN with bevacizumab-800CW. Furthermore, although
micrometastasis are associated with poor prognosis, it is yet unknown whether
the treatment of micrometastasis with adjuvant chemotherapy will result in any
clinical benefit. However, the most recent Dutch guidelines advice the use of
adjuvant chemotherapy in this *upstaged* group and thus will receive adjuvant
chemotherapy.

The potential benefits or harms for the patient are based upon the difference
in staging compared to standard staging techniques. With negative lymph nodes
after H&E, additional ultrastaging techniques will be performed. If
micrometastases are detected in the lymph nodes this will not result in any
clinical consequences, due to the lack of evidence for effectivity of adjuvant
chemotherapy in these patients. However, if ultrastaging techniques result in
detection of lymph nodes with macrometastases, these patients will be offered
adjuvant chemotherapy. Furthermore, aberrant lymph nodes will be excised and
analysed as the other lymph nodes. Potentially resulting in treatment with
adjuvant chemotherapy, while, this would not be given if the aberrant node
would not be excised.

Since patients will receive an additional colonoscopy, preceded by an
additional MBP, patient have an additional risk of complications associated
with MBP and colonoscopy. Risks associated with MBP that have been described
are hypovolemia, electrolyte imbalances, renal failure and discomfort for the
patient. Furthermore, risks associated with colonoscopy that have been
described are perforations and post-colonoscopy bleeding. However, reported
risks have shown to be low with 0.05% and 0.98% for perforations and
postcolonoscopy bleeding respectively. More importantly, these complications
are mostly related to polypectomies and would be resolved immediately since
injection is performed during surgery. The risk of comparable procedures such
as endoscopic tattooing is low (<0.22% complication rate) and are routinely
performed. Bevacizumab-800CW has a low-risk safety profile, see for further
elaboration (Chapter 11 of study protocol: Potential issues of concern). Since
we will only inject a small amount of tracer, we expect these risks are
negligible.

Consequently, the potential benefit of this study is a potential better
oncological outcome for patients who will be offered adjuvant chemotherapy
since ultrastaging techniques delivered micrometastases, who would otherwise
not be found. Potential risk of this study is the risks associated with MBP and
colonoscopy.