A pilot study to assess the safety and feasibility of fluorescent sentinel lymph node identification in colon carcinoma using intravenous 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.

Recently, more research is conducted in tumour-targeted tracers as they have
many advantages compared to ICG. For example, tumour-targeted tracers can be
preoperatively administered which aid logistics, binds to tumour and
metastases, thus allowing more time for uptake in patients with larger tumours
and lymph node metastases. These properties may result in increased accuracy
and would be more broadly applicable compared to ICG. Furthermore,
tumour-targeted tracers can also be administrated intravenously and potentially
identify lymph node metastases without having to perform a colonoscopy.
However, administration via colonoscopy of tumour-targeted tracers for the
detection of lymph node metastases in colon carcinoma has not been performed
yet, and intravenous administration would be a step after administration via
colonoscopy.
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 intravenous administration.

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, 15mg bevacizumab-800CW is intravenously
administered. The patient is observed during one hour after injection of
bevacizumab-800CW.
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. Histological section of 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.

The potential benefits or harms of the patients are based on the difference in
staging that could potentially be an effect of the ultrastaging techniques. If
macrometastases or micrometastases are detected during ultrastaging techniques,
patients will be given adjuvant chemotherapy, according to the Dutch Guideline
Colorectal Carcinoma. A potential benefit of this study could be that patients
receive adjuvant chemotherapy, due to outcomes of ultrastaging techniques,
while they would not receive adjuvant chemotherapy if regular staging
techniques were used. 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.
In this study, safety data related to (the administration of)
bevacizumab-800CW, a targeted optical fluorescent tracer, will be collected and
evaluated. VEGF-A (Vascular Endothelial Growth Factor-A) is highly upregulated
in tumour tissue of colorectal origin (UMCG data set: (n=35), 100%) and can be
targeted by using bevacizumab (Avastin®, Roche). Bevacizumab is a recombinant,
high affinity, humanized IgG1 monoclonal antibody with specific affinity for
VEGF-A.
The infrared dye IRDye800CW (LI-COR Biosciences, Lincoln, NE, USA) is a
fluorescent dye applicable for clinical use, produced by REGIS technologies.
Conjugation of the fluorescent dye to bevacizumab, purification and formulation
will be performed at the department of hospital and clinical pharmacy of the
UMC Groningen to create bevacizumab-800CW. The new tracer bevacizumab*
IRDye800CW has been evaluated preclinical in tumour bearing nude mice and an
extended single microdose toxicity study has been performed by NOTOX, which did
not show toxicity (more information can be found in the IMPD).
Several studies using bevacizumab-800CW (see ClinicalTrials.gov NCT02113202 ,
NCT01972373 , NCT02129933 , NCT01508572 , NCT03913806 , NCT03877601 ,
NCT04212793 , NCT03620292 ) or other targeted fluorescent agents (see
ClinicalTrials.gov NCT01987375, NCT02415881 ) have received regulatory
approvals to administer the non-FDA approved targeted fluorescent agents to
humans.
Five studies using bevacizumab-800CW in a total of 91 patients have been
completed. In 82 of the 91 patients a dosage of 4.5-50mg bevacizumab-800CW was
administered, the remaining nine patients received topical bevacizumab-800CW .
Adverse events were observed in only one study. The study used systemic and
topical bevacizumab-800CW for guidance during endoscopic mucosal resection.
Four adverse events were observed in 14 patients, all adverse events are known
complications of endoscopic mucosal resection: bleeding (n=2), nausea/headache
(n=1) and fever (n=1). Thus, most likely these events were not tracer-related .
The time investment of participants is considered reasonable. The visit for the
tracer administration visit will take around 2 hours total.