Fluorescein for Lymphatic Mapping and Sentinel Lymph Node (SLN) 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 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.

Inclusion Criteria:

1. Ability to provide informed consent and have signed an approved consent form that
conforms to federal and institutional guidelines.

2. Between 18 and 90 years of age.

3. Have a primary melanoma that is cutaneous (including head, neck, trunk, extremity,
scalp, palm, sole, subungual skin tissues)

4. Have a primary melanoma meeting one of the following criteria:

1. Primary melanoma was ≥ 0.75 mm Breslow thickness and Clark level III or

2. Primary melanoma was Clark level IV/V or

3. Primary melanoma was ulcerated or

4. Primary tumor mitotic >1/mm2 or

5. Primary melanoma was less than 0.75 mm Breslow thickness with one or more poor
prognostic features (regression > 75%, vertical growth phase, mitotic Count >
1/mm2, transected deep biopsy margin) or

6. Have had a prior excision (non-wide local excision) of a melanocytic lesion with
development of a primary melanoma in the excision scar or

7. Have had a wide locale excision within the past 120 days of a primary melanoma as
defined in (a-f) above but not yet undergone a SLNB

5. Clinically negative lymph nodes.

6. ECOG performance status 0-1

Exclusion Criteria:

1. Primary melanoma of the eye, mucous membranes or internal viscera.

2. Physical, clinical, radiographic or pathologic evidence of satellite, in-transit,
regional or distant metastatic disease.

3. Skin grafts, tissue transfers or flaps that have the potential to alter the lymphatic
drainage pattern from the primary melanoma to the lymph node basin.

4. Allergy to radiocolloid or fluorescein.

5. Inability to localize 1-2 SLN drainage basins via lymphatic mapping. (e.g., no basin
found which emits gamma-radiation after injection with technecium-99m or more than 2
basins are found which emit gamma-radiation.)

6. Prior completion lymph node dissection or SLNB that may have altered the lymphatic
drainage from the primary cutaneous melanoma to a potential lymph node basin.

7. Organic brain syndrome or significant impairment of basal cognitive function or any
psychiatric disorder that might preclude participation in the protocol, or be
exacerbated by therapy.

8. Melanoma-related operative procedures not corresponding to criteria described in the
protocol.

9. Primary or secondary immune deficiencies or known significant autoimmune disease which
would pose a risk to the participant based on the physician's judgment.

10. History of organ transplantation.

11. Pregnant or lactating women.

12. Participation in concurrent experimental protocols or alternative therapies that might
confound the analysis of this trial. Adjuvant therapy protocols after recurrence are
acceptable.

13. Nonmalignant systemic disease (e.g., cardiovascular, renal, hepatic, etc.) that
precludes a patient from being subjected to any of the treatment options or that would
prevent prolonged follow-up based on the physician's judgment.