Trial Information

Fluorescein for Lymphatic Mapping and Sentinel Lymph Node Biopsy in Patients With Stage I and II Malignant Melanoma

OBJECTIVES:

The primary goal of this study is to evaluate the ability of intradermal fluorescein to
detect sentinel lymph nodes (SLNs) in patients with stage I and stage II melanoma.

Primary Objectives To determine the co-localization between fluorescein and technetium-99m
labeled sulfur colloid in SLNs.

Secondary Objectives

1. To evaluate the ability to detect fluorescein fluorescence transdermally with a
fluorometer.

2. To evaluate if there is a correlation between SLN ex-vivo fluorescent intensity and SLN
ex-vivo gamma radiation intensity.

3. To evaluate if there is a correlation between SLN ex-vivo fluorescent intensity and SLN
metastasis.

4. To evaluate if fluorescein fluorescence can be detected in the SLN after fixation and
histological processing.

5. To evaluate the toxicity and safety of intradermal fluorescein injections.

The management of regional lymph nodes in patients with clinically localized primary
melanomas has been controversial. An elective lymph node dissection at the time of removal
of the primary melanoma has been favored by many. The proponents of elective lymph node
dissection has based their opinion of the hypothesis that melanoma spreads in an orderly
fashion from the primary site to regional lymph nodes and then systemically. Thus early
removal of lymph node tumor deposits may prevent subsequent systemic dissemination.1-7 Four
prospective randomized trials of elective lymphadenectomy have tested this hypothesis.8-11
In all of these trials, elective lymphadenectomy did not result in a significant survival
benefit. In one of the trials8, a subgroup analysis indicated that elective lymphadenectomy
may benefit patients younger than 60 years of age, especially those with nonulcerated
primary melanomas and melanomas between 1-2mm in thickness. Based on these results,
elective lymphadenectomy for patients with stage I and II melanoma is not advocated and this
has resulted in a more selective evaluation of the regional lymph nodes and development of
the sentinel lymph node biopsy (SLNB) technique.

The sentinel lymph node (SLN) concept is based on the hypothesis that tumor cells from
primary melanomas metastasizes through the lymphatic system to regional lymph nodes in an
orderly fashion and that mapping of the lymphatic system can identify the first or
"sentinel" lymph node to receive metastatic tumor cells. This sentinel lymph node will
become involved with metastasis before any other node in the regional lymph node basis and
if involved will reflect the pathologic status of the entire regional nodal basin. Morton et
al.12 were the first to evaluate the SLN concept in patients with stage I melanoma. In this
study of 237 lymph node basins in 233 patients, the SLN was identified 82% of the time and
it predicted the pathologic status of the nodal basin in 99% of cases. Since this
preliminary study, substantial progress has been made improving and standardizing the
techniques for lymphatic mapping and SLNB.

Use of a vital blue dye such as 1% isosulfan blue (Lymphazurin®) has been part of the
lymphatic mapping and SLNB since its introduction. At the time of operation, 3-5 ml of the
vital blue dye is injected intradermally around the intact primary melanoma or the tumor
biopsy site. The dye rapidly diffuses into the lymphatic system and is carried by afferent
lymphatic trunks to the SLN. An incision is made over the draining nodal basin and the blue
afferent lymphatic channels are followed to the first draining lymph node(s), the sentinel
lymph nodes. With the use of a vital blue dye, the SLN can be identified in approximately
87% of cases.13 This leaves 13% of patients unable to benefit from a SLN evaluation.
Gershenwald et al. demonstrated that SLN identification improved from 87% to 99% when
technetium-99m labeled sulfur colloid was combined with the vital blue dye. 13 To increase
the detection rate of SLNs, two additional techniques are commonly used: a) pre-operative
lymphoscintigraphy using a technetium-99m labeled sulfur colloid or human albumin
radiotracer 14, 15 to better delineate the lymphatic drainage and identify multiple drainage
basins and b) intraoperative use of a handheld gamma probe to better localize the SLN.
Currently, using the vital blue dye technique in combination with a radiotracer identifies
the SLN in up to 99% of cases.13, 16, 17 Based on these findings, most clinicians now
recommend using a combined modality approach which is considered the "gold standard" for SLN
localization in patients with primary melanoma. Although the technetium-99m labeled sulfur
colloid adds a greater detection ability, formal studies have not been reported using this
alone. Informal observation finds that one can pick up radioactivity in nodes which are not
blue more often than one picks up blue nodes that are not radioactive, but again the ideal
situation is to be able to use two tracers at once.

Although 1% isosulfan vital blue dye increases the detection of SLNs when combined with a
radiotracer, it has several drawbacks. First, the dye can diffuse throughout the operative
wounds making dissection and SLN identification difficult. This is especially concerning if
the afferent lymphatic channels are cut. Second, 1% isosulfan blue dye has been associated
with an anaphylactoid reaction or a life threatening anaphylactic shock in 0.1 - 2% of
patients undergoing lymphatic mapping and SLNB.18-23 Third, a recent shortage in 1%
isosulfan blue has resulted in a decreased access to the compound for patients and
clinicians. Thus, there is a great need to develop new lymphatic mapping and SLN
identification techniques.

Fluorescein is an orange-red powdered compound, designated by the formula C20H12O5, which
exhibits intense greenish-yellow fluorescence in alkaline solution. It has been used
extensively in surgery and medicine for decades for diagnostic purposes. Topical fluorescein
is routinely used in ophthalmology to assess corneal lesions.24 Intravenous fluorescein is
used in vascular surgery to measure vascular perfusion25 and in skin and melanoma surgery to
assess the viability of skin flaps.26, 27 Intradermal fluorescein injections have been used
to identify pedal lymphatics to facilitate lymphangiography.28 This study was designed to
look at both the safety and efficacy of using 10% fluorescein mixed 1:1 with 1% lidocaine
hydrochloride. Cooper et al. reported on intradermal injection of fluorescein in 1,047
patients without adverse reactions.28 In this study, "not a single immunologic reaction was
identified. In two patients, local skin sloughing occurred at the injection site due to
inadvertent administration of a 3;1 fluorescein-lidocaine mixture rather than the prescribed
1:1 ratio". Dan et al.29 used intramural bowel injection of fluorescein in 120 patients
with colon cancer to map the lymphatics in patients with colon cancer. Fluorescein was able
to identify the sentinel lymph node in 97% of patients and none of the 120 patients suffered
any adverse reactions. We have previously shown that Cy5-cobolamine bioconjugate injected
intradermally into the hind limb of pigs is able to identify inguinal sentinel lymph
nodes.30 More recently, we have also determined that fluorescein injected intradermally into
the limb of pigs is also able to identify the sentinel lymph node. Additionally, when 1%
isosulfan blue is injected in the same location as fluorescein, the two detection techniques
co-localizes in the afferent lymphatics and the sentinel lymph node. The fluorescent signal
from fluorescein provides improved detection of the afferent lymphatic and the sentinel
lymph node compared to 1% isosulfan blue. Moreover, fluorescein fluorescence is clearly
visualized transdermally and enables an improved localization of the sentinel lymph node
prior to performing a skin incision. (Andtbacka RH, McGreevy JM, Grissom CB et al.
unpublished results). This transdermal fluorescence may enable elimination of the
radiotracer in sentinel lymph node detection. Based on these findings we are now proposing a
phase I/II clinical trial in melanoma to assess the safety and feasibility of intradermal
fluorescein in the detection of SLNs in patients with stage I and stage II melanoma.