Melanoma lymphoscintigraphy and lymphatic mapping

Melanoma Lymphoscintigraphy and Lymphatic Mapping Claudia G. Berman, Junsung Choi, Maria R. Hersh, and Robert A. Clark Lymphoscintigraphy in melanoma has proven to be a reliable method to identify regional lymph nodes a t risk for metastases. The first lymph node to drain a cutaneous lesion, the sentinel lymph node (SLN), is predictive of the metastatic status of the regional lymph node group. Lymphatic mapping allows for the identification of the SLN and for selective lymph node sampling. Selective lymph node sampling is less inva-

sive and because only a small quantity of high-risk tissue is submitted for pathological examination, it allows for a more complete and comprehensive pathological examination, which identifies melanoma with up to 100 times the sensitivity of conventional examinations. Copyright 9 2000 by W.B. Saunders Company

YMPHOSCINTIGRAPHY is the injection of radioactive particles that are then imaged as they pass through afferent lymphatic vessels to their respective lymph node drainage basins. Lymphoscintigraphy is receiving increasing interest as a means to stage regional lymph nodes in early clinical stage melanoma. It promises to both simplify and improve the accuracy of staging these patients. In its first successful modem application, lymphoscintigraphy has been applied to the localization of the true nodal drainage basins of melanomas situated in regions of highly variable drainage, in particular, the trunk, head, and neck. t-4 This allows the surgeon to perform a relevant prophylactic lymphadenectomy and to choose patients for adjuvant alpha-2b-interferon adjuvant therapy based on a sample of lymph nodes truly at risk. 5,6 There has been a rapid increase in the incidence of malignant melanoma. The lifetime risk for an American is expected to surpass 1% within the next few years. This trend will probably continue indefinitely given the increasing exposure of fair-skinned populations to intense sunlight environments. Thin, early-stage melanomas are usually cured by local excision. Lymphatic spread from the primary site can result in dissemination and death. The probability of this scenario becomes distinctly greater when primary lesions have surpassed 1 mm in thickness and it is highly likely to occur for primary lesion thicknesses greater than 4 mm. It is in these groups of patients, those with a meaningful risk of death from disseminated disease but no clinical proof of dissemination, that lymphoscintigraphy is becoming a necessary and powerful tool.

( > 1.5 ram) primary tumors, Stage III: disease with regional spread to skin more than 5 cm from the primary tumor or to the regional lymph nodes, and Stage IV: patients with distant metastases. Patients with thinner Stage I lesions have a 5-year survival rate of better than 95%, compared with a survival rate below 70% for the thicker Stage II lesions. The 5-year survival rate drops to approximately 30% in patients with resected Stage Ill regional disease and is anecdotal in patients presenting with metastatic disease. Two pathological conventions, the Clark's level and Breslow thickness, have been used to assign lesions within and between Stage I and Stage II. These schemes attempt to differentiate thinner from thicker primary-only disease. Clark's levels I through V range from in situ disease (I) to subcutaneous invasion (V). Level III describes a primary, which fills the papillary dermis and abuts, but does not invade, the reticular dermis. Although Clark's system has been shown to correlate well with clinical outcomes it has been largely superceded by Breslow thickness, which uses an ocular micrometer to measure the depth of the lesion's penetration below the granular layer of the skin. According to the Breslow system, a T I tumor is less than or equal to 0.75 mm and a T2 tumor is between 0.76 and 1.50 mm. TI and T2 tumors together constitute Stage I disease. T3 lesions range from ! .51 to 4 mm and T4 lesions exceed 4 mm in thickness. The grouping ofT3 and T4 lesions corresponds to Stage II melanoma. The Breslow system has been found to be simpler, more reproducible, and better predic-

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MELANOMA STAGING

The generally accepted staging for malignant melanoma involves Stage I: denoting thinner ( < 1.5 mm) primary tumors, Stage II: denoting thicker

From the H. Lee Moffttt Cancer Center, Department of Radiology, Tampa, FL. Address reprint requests to Claudia G. Berman, MD, 1-1.Lee Moffitt Cancer Center, Department of Radiology, 12902 Magnolia Drive, Tampa, FL 33612. Copyright 9 2000 by W.B. Saunders Company 0001-2998/00/3001-0008510. 00/0

Seminars in Nuclear Medicine, Vol XXX, No 1 (January), 2000: pp 49-55

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tive of outcomes than the Clark levels. A rough correspondence exists between T1, T2, T3, and T4 as defined by the Breslow system, and Clark levels II, III, IV, and V, respectively. Approximately 85% of patients with malignant melanoma will present with Stage I or Stage II disease, as defined earlier. A substantial proportion of these patients harbor inapparent nodal and perhaps visceral disease. The recognition of nodal disease upstages the patient with important implications for prognosis and, according to recent data, treatment. It is of the utmost importance, therefore, that the lymph nodes draining the primary site be accurately identified and sampled. SENTINEL LYMPH NODE CONCEPT

The sentinel lymph node (SLN) is the first lymph node encountered by the lymphatic channel draining the tumor (Fig 1). It has been proposed, and appears to have been proven, that in melanoma the pathological status of the SLN accurately predicts the status of the entire nodal basin. 7 Not only nodes of the draining basin but in-transit lymph nodes, situated between the injection site and the anatomically recognized regional lymph node groups, have been found to be SLNs and to accurately predict the pathological status of the regional nodal basin as a whole. Strong support for the SLN hypothesis has

Fig 1. An anterior chest lymphoscintigram shows 2 separate afferent lymphatic vessels that each travel to the same single SLN (arrow).

been shown in both melanoma and, more recently, carcinoma of the breast. 8,9 Giuliano et al 1~ was the first to use a vital blue dye to identify SLNs for tumor staging. They obtained an intraoperative lymphogram and SLN identification in melanoma patients. Our group at H. Lee Moffitt Cancer Center and Research Institute at the University of South Florida, as well as other investigators, have combined the use of radiocolloid injection and vital blue dye for lymphatic mapping. We have found the combination of the 2 approaches complementary in melanoma as well as in carcinoma of the breast.11.a2 The ability to improve the diagnostic accuracy of nodal staging while at the same time reducing the morbidity of nodal dissection of clinically node-negative basins is highly appealing for patients with early stage melanoma or breast cancer. 13-15 For these techniques to be useful, there are 2 biological requirements. First, the lymphatic drainage identified by lymphoscintigraphy must identify the nodal basins, as well as the the specific nodes within the basins, that receive lymphatic flow from the lesion. Second, the pathological status of the first lymph nodes into which these lymphatics drain, the SLN, must be predictive of the status of the remainder of the basin and there must be no false-negative SLN or skip metastases. 16 The reliability of vital blue dye, radiocolloid lymphoscintigraphy, and lymphatic mapping for accurate identification of the true physiological nodal drainage basins of melanoma skin lesions, in distinction from the anatomically presumed nodal drainage basins, has been established from the experience of our department at H. Lee Moffitt Cancer Center and Research Institute, as well as from other centers. In particular, we have showed a discordance rate between the true physiological drainage based on lymphoscintigraphy and lymphatic drainage based on the classical anatomically presumed nodal drainage basins of approximately 40% for truncal, head, and neck lesions with the head and neck discordance rate approaching 70%. 17 We have yet to see a single initial nodal relapse in a lymph nodal basin not previously identified by lymphoscintigraphy as the primary drainage basin among more than 400 of our patients, with a mean follow-up of 5 years. 18 Reliance on the SLN, the first node within the basin receiving lymphatic flow from the primary site, as the arbiter of the status of the remainder of

LYMPHOSCINTIGRAPHY AND LYMPHATIC MAPPING

the nodes within the basin postulates an orderly progression of metastasis requiring, first, involvement of the SLN. Our experience, as that of others, of the accuracy of the SLN in predicting the pathological status of the remaining nodes of the basin is compelling. A prospective multi-institutional collaborative study of 42 patients with primary melanomas thicker than 0.75 mm and shown on lymphoscintigraphy to have only 1 primary draining nodal basin were studied) 6 The mean tumor thickness was 2.2 mm. Approximately 1 mCi (3.7 X 107 Bq) 99m-Technetium tagged human serum albumin (HSA) was injected intradermally in 4 equal aliquots surrounding the primary site. Scanning was performed immediately and 2 hours after injection. Only 1 nodal basin served as first echelon drainage for the primary lesion. The hot area on the initial scan was tattooed and subsequently served to mark the site for surgical incision to harvest the sentinel node. Either or both the maximum focus of sequestration of radionuclide activity or the entry of a blue-stained afferent lymphatic leading from the primary site into the node was used to identify the SLN. The harvesting of the sentinel node was followed up by a formal lymph node dissection in all patients. Head and neck primary sites were excluded from the study. Metastatic disease was present in 8 of 42 patients (nodal basins). The SLN was the only site of metastatic disease in 7 of these 8 patients. Detailed statistical analysis (beyond a priori common sense) shows this to be a highly significant finding. No false-negative sentinel node studies (skip metastatic nodes) occurred. This early study has been corroborated by substantial subsequent experience. PATHOLOGICAL EXAMINATION

The promised improved sensitivity of this method stems not only from the identification of the true SLN at risk but from the opportunity this provides for the pathologist to bring to bear a more intensive examination on this maximum-risk lymph node. This is of particular importance in that roughly one quarter of patients with histologically negative lymph node basins will progress to metastatic disease within 5 years of nodal dissection. Standard histological techniques probably understage a significant proportion of patients. It is estimated that less than 1% of the submitted material is examined by standard histological examination of lymph nodes, comprised of sectioning of

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the nodes through the middle and staining only 1 or 2 sections with standard dye stains. The standard approach permits the identification of 1 melanoma cell against a background of 10,000 lymphoid cells. Relieved of the need to stain and examine the large harvest of lymph nodes delivered by a standard nodal basin dissection, the pathologist is able to serially section the 1 or 2 true high-risk nodes. This allows for a more thorough examination of a much smaller volume of higher risk tissue. Additionally, the more sensitive (and specific) immunohistochemical stains can be used in addition to standard dye stain techniques. It is estimated that by adding melanoma-specific immunohistochemical staining to serial sectioning of the nodes, an entire order of magnitude can be added to the sensitivity of the pathological examination, allowing for the identification of 1 metastatic malignant melanoma cell against a background of 100,000 normal lymphoid cells. A cell culture technique performed in tandem with standard hematoxylin and eosin examination at H. Lee Moffitt Cancer Center is being performed to further increase the sensitivity of the pathological examination of these key nodes. A 31% upstaging from Stages I or II to Stage III occurred among cell-cultured specimens of histologically negative nodes. Additionally, this upstaging correlates with an increased risk of subsequent recurrent disease. Reverse transcription-polymerase chain reaction (RT-PCR) is an approach that takes advantage of the high specificity of tyrosinase messenger RNA for melanocytes. It is presumed that the presence of this messenger RNA in cellular material from a lymph node draining a melanoma is tantamount to a diagnosis of lymph node metastasis. The RT-PCR assay greatly enhances the sensitivity of the pathological examination and is a quick and reproducible technique. Our group has performed a comparative study pitting RT-PCR against standard dye stain techniques, testing SLN samples from 29 patients with intermediate thickness melanomas. Standard dye staining techniques identified melanoma metastasis to the SLN in 38% of patients. RT-PCR assay identified melanoma metastasis to the SLN in 66% of patients, including all patients identified by standard staining techniques.~8 We have been able to correlate clinical outcomes to RT-PCR SLN results. 19 Among 74 patients studied, 14 patients were node positive by both

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routine histology techniques and RT-PCR analysis techniques and experienced a 3-year recurrence rate of 42%. Thirty-three patients were node negative by both techniques and experienced a 3-year recurrence risk of 6.6%. Twenty-seven patients fell into the group with metastatic adenopathy in SLNs identified only on the more sensitive RT-PCR assay. These patients experienced a 3-year recurrence risk of 22%, intermediate as would be expected, between the groups with higher and lower microscopic tumor burden. These findings correlate with our estimation of sensitivity for RT-PCR for identifying 1 metastatic melanoma cell against a background of approximately 1 million nodal lymphoid cells, a 2 order of magnitude improvement in sensitivity from that achievable with standard histological techniques practical with standard lymph node basin dissection specimens. The development of lymphoscintigraphy and SLN examination has coincided with 2 landmark developments in the therapy of early stage melanoma. Perhaps the more significant of these is the demonstration of increased survival in Stage III (regional node positive) and possibly Stage IIb (tumor skin depth greater than 4 ram) melanoma patients when high doses of alpha-2b-interferon were given as adjuvant postoperative systemic treatment (Eastern Cooperative Oncology Group [ECOG 16841). This survival advantage was more clear cut for Stage III than for Stage IIb patients, which may be attributable to the relatively small sample size of patients in the study with Stage IIb disease. There was a 42% increase in the continuous relapse-free survival rate at 5 years from 26% to 37% in those patients that received alpha-2binterferon. There was also an improvement in overall survival from 2.8 years in patients randomized to not receive biotherapy compared with 3.8 years in those patients receiving alpha-2b-interferon and this was statistically significant. When only patients with nodal metastases (Stage III) are considered, the P values for disease-free survival and overall survival are .006 and .0006, respectively.2~ ECOG 1684, which opened in 1985, did not use lymphoscintigraphy or SLN examination. One can only speculate how the improved accuracy and sensitivity provided by lymphatic mapping with augmented pathological examination of the SLN will impact on the clinical results that have been achieved. If the assumption is made that imperfect

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adjuvant therapy is more useful in patients with smaller subclinical tumor burdens, it can be hoped that patients whose disease is detectable only by SLN examination will benefit even more dramatically than those node-positive patients who participated in ECOG 1684. The second landmark study deals with the therapeutic benefit of elective lymph node' dissection (ELND) in patients with intermediate thickness melanomas, generally those 1 to 4 mm in thickness. A succession of randomized studies has failed to show any survival advantage to ELND in patients with intermediate thickness, clinically node-negative melanoma. It had been logically anticipated that this group would benefit from such therapy in that they show a high risk for initial regional relapse rather than distant metastatic failure. The recently reported Intergroup Melanoma Trial is the first randomized trial concentrating on this group of clinically node-negative patients with high risk for regional relapse, which has suggested a benefit from ELND. It is also the first to use lymphoscintigraphy to assure that the lymph node basins removed in fact are those that drain the primary lesion. Clinical benefit was particularly apparent for patients younger than 60 years of age with primary lesions 1.1 to 2.0 mm thick: Five-year survival rates in these patients improved from 84% in those not receiving ELND to 96% in patients receiving ELND (P = .007). 2I TECHNICAL FACTORS IN LYMPHOSCINTIGRAPHY

Modern lymphoscintigraphy is usually performed with technetium-99m, which offers many advantages over alternative radioisotopes. Worldwide, there are several choices of commercially available particles to label with technetium-99m. A comparison between sulfur colloid, colloidal HSA, and antimony trisulfide colloid showed that all 3 agents showed comparable distribution patterns. 22 However, there may be some difference in the degree of uptake and in the number of lymph nodes routinely visualized. Colloidal HSA and noncolloidal HSA are commonly used for lymphoscintigraphy because of its uniform particle size. However, HSA, particularly in noncolloidal form, seems to pass through the lymph nodes rapidly. Instead of uptake in 1 or 2 SLNs, 6 to 8 lymph nodes with radioactive uptake are commonly identified after the injection of albumin. It is also more difficult to

LYMPHOSCINTIGRAPHY AND LYMPHATIC MAPPING

use the hand-held gamma probe and identify SLNs when there are a number of lymph nodes that are radioactive. Because the albumin passes through the lymph nodes so rapidly, there is also a concern that it might pass through the SLNs by the time the patient is presented to the operating room, thus defeating the purpose of SLN mapping. Technetium antimony trisulfide colloid has favorable imaging and mapping characteristics for lymphoscintigraphic studies because its particle size, 3 to 30 nm, is optimal for transit through lymphatics and localization in nodes without phagocytosis. Unfortunately, antimony trisulfide colloid is no longer produced for use in the United States. It is still available in Australia, Canada, and Europe and is used widely there as is rhenium colloid. Hung et a123have produced acceptable lymphoscintigraphic images with technetium sulfur colloid filtered to a maximum particle size of 100 nm. In practice, nearly all of their filtered activity was distributed in the 15 to 50 nm range, z3 At H. Lee Moffitt Cancer Center and Research Institute, sulfur colloid is used after filtration with a 0.2-micron (20 nm) filter. It is important that the sulfur colloid be fresh and used less than 2 hours after preparation because over time there is clumping of the colloidal particles, which in effect causes them to be larger in size and less effective. The 0.2-micron-filtered sulfur colloid preparation appears to work very reliably, consistently identifying 1 or 2 SLNs for excision. Alazraki et a124 have systematically studied the duration of heating and elution time, in addition to filtration in an effort to truly optimize the particle size distribution of the sulfur colloid injectate. Of note, we and others have had comparable, excellent results in our melanoma studies with less rigorously prepared agents.

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melanoma. One mL of diluent is divided into 4 equal aliquots. The dose is placed in tuberculin syringes and injected intradermally, raising skin wheals about the periphery of an intact lesion. In those cases after biopsy, 2 injections are made on each side of the scar but not within the scar. Each injection is about 1 cm from the scar. It is important not to inject at the ends of the scar as this would lead to more disparate drainage than the intact lesion and will not necessarily reflect the drainage pattern of the original tumor. A caution generally pertinent to the use of lymphoscintigraphy for SLN identification relates to the importance of confirming that the lymph nodes that are identified are indeed SLNs. The nuclear physician must track the afferent drainage channels to see if multiple drainage channels culminate in multiple SLNs or if the lymphatic channels converge to terminate in a single SLN. One must also ascertain whether or not the visualized lymph nodes run in series, meaning that the first lymph node is the sentinel node, or in parallel, meaning that each imaged lymph node represents a SLN (Fig 2).

LYMPHOSCINTIGRAPHY PROTOCOL

In most cases of melanoma involving the trunk and extremities, 450 ~tC (1.67 x 107 Bq) of technetium is used. The dose is decreased to 250 pC (9.25 X 106 Bq) in procedures involving the head and neck and in those patients in which a lesion is very near the drainage basin. The dose is decreased because there may be less artifact from the injection site when a lower dose is used. False-negative lymph nodes can be caused by masking the lymph nodes near the injection site by injection site artifact. A very high specific activity is used in

Fig 2. The first lymph node (arrow) in this left lateral neck lymphoscintigram is the SLN, although 3 lymph nodes are visualized.

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After injection, the patient is immediately placed under a standard large field of view gamma camera using a high resolution collimator and a 10% imaging window at the 140 keV technetium energy peak so that the afferent drainage can be identified. Cutaneous lymphatic flow is so rich that afferent lymphatics are immediately seen and can be traced in most cases. The camera will be positioned over the regional lymph node groups that drain the lesion and an attempt is made to localize the SLNs. This is performed by following the afferent channels until they persistently accumulate in a particular focus. Cine imaging is quite useful in melanoma cases because the lymphatic flow is rich and progression to the regional lymph node basins can occur quite rapidly. Dynamic imaging is an essential element in SLN identification. 25 The patient is imaged in multiple projections for localization. Using the hand-held gamma probe is an effective way to accurately localize lymph nodes. It also allows for the marking of the lymph nodes in the same anatomic position that will be used in the operating room. An attempt is made to find intransit lymph nodes, those lymph nodes between the injection site and the regional lymph node group which, by definition, are SLNs. Lymphoscintigraphy is the only way to find these lymph nodes. Delayed imaging is very rarely used. Once the lymph nodes are localized, the skin can be marked with an indelible pen or a tattoo. The body contour and body landmarks can be outlined using a technetium marker. Another technique involves the insertion of a cobalt flood source between the patient and the detector (Fig 3). The patient's soft tissues attenuate the gamma rays allowing for the identification of various body landmarks. Technical failures are very rarely seen (less than 5% of injections). Usually, technical failures are seen in patients in which there is severe induration after an excisional biopsy. If there is no discernable lymphatic flow after 40 minutes the patient is reinjected with the same dose of radiopharmaceutical, but the injections are placed farther from the epicenter of the scar, about 1.5 cm from the scar. Massage and warm compresses can be applied around the injected area to stimulate lymphatic flow. If there is no discernable lymphatic flow after an additional 45 minutes of imaging, the patient is asked to return after a 2- to 3-hour delay. The study can usually be recovered by this point. However,

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Fig 3. Whole body Iocalizer lymphoscintigram uses a cobalt flood source to define the body contour,

there have been cases where there was no discernable lymphatic flow even after delayed imaging. Lymphatic flow and SLNs are usually identified if a patient returns for a second study the next week. The amount of the radiopbarmaceutical trapped in the lymph nodes increases over time. It is recommended that the injections be performed at least 2 to 4 hours before surgery. This gives sufficient time for enough of the radioactive particle to be trapped in the lymph nodes to provide good target-to-background ratios without any prob-

LYMPHOSCINTIGRAPHY AND LYMPHATIC MAPPING

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l e m r e l a t e d to the d e c a y o f the t e c h n e t i u m . T h e r e is a g r e a t deal o f latitude as to w h e n t h e s u r g e r y c a n b e p e r f o r m e d a n d surgeries c a n b e u n d e r t a k e n i m m e d i a t e l y a f t e r i n j e c t i o n a n d e v e n the d a y a f t e r an injection. CONCLUSION

T h e m o s t useful a n d c o s t - e f f e c t i v e i n t e g r a t i o n o f t h e s e n e w d i a g n o s t i c a n d t h e r a p e u t i c tools will

f u r t h e r b e e l u c i d a t e d in c l i n i c a l studies n o w in p r o g r e s s a n d o t h e r s s o o n to follow. It is i m p o r t a n t to stress m e t i c u l o u s p l a n n i n g a n d t h o r o u g h c o o p e r a tion a m o n g t h e r e l e v a n t d e p a r t m e n t s ( n u c l e a r m e d i cine, surgery, a n d p a t h o l o g y ) to a s s u r e that t h e m a x i m u m p o t e n t i a l o f t h e s e i n n o v a t i o n s is q u i c k l y and consistently achieved for the benefit of our p a t i e n t s a n d f o r r e l i a b l e e x e c u t i o n o f c l i n i c a l studies.

REFERENCES

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