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From the Cutaneous Oncology Program, Lakeland Regional Cancer Center, Lakeland, Florida.
Correspondence: Address correspondence and reprint requests to: Douglas Reintgen, MD, Lakeland Regional Cancer Center, 3525 Lakeland Hills Blvd., P. O. Box 91057, Lakeland, FL 33804–1057; Fax: 863-904-1802; E-mail: Doug.Reintgen{at}lrmc.com
ABSTRACT
Lymphatic mapping and sentinel lymph node (SLN) biopsy have changed the standard of care for patients with malignant melanoma, by providing a less morbid procedure to obtain the nodal staging information that is critical for therapeutic decisions. Detailed examination of the SLN identifies patients who have an increased risk for recurrence and death. Patients whose melanoma is upstaged with very sensitive assays based on reverse transcriptase polymerase chain reaction technology are better targeted for clinical trials or surgical or adjuvant therapies. In the future, melanoma may be "ultrastaged" by examining the SLNs, peripheral blood, and bone marrow. This may improve identification of patients who are surgically cured of their disease and therefore can be spared the side effects of more radical surgery or the toxicities of adjuvant therapy. The lymphatic mapping procedure is the most accurate way to determine the tumor status of the regional lymph nodes.
Key Words: Lymphatic mapping • Malignant melanoma • Sentinel lymph node biopsy • Staging
The presence or absence of lymph node metastases in patients with solid tumors is the most powerful factor for predicting recurrence and survival.1 The American Joint Committee on Cancer (AJCC) Melanoma Staging Committee organized a database of more than 17,000 melanoma patients with a mean follow-up of 5 years.2 This powerful database identified important prognostic factors based on the primary melanoma (Table 1). Because lymph node status dwarfs the importance of any prognostic factor based on the primary tumor (Table 2), it makes sense to concentrate on more accurate methods to stage the regional nodes of patients with malignant melanoma.
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Lymphatic mapping with sentinel lymph node (SLN) biopsy allows the surgeon to identify the node most likely to contain metastases.4 This significantly reduces the morbidity of the cancer operation and the quantity of nodal tissue that the pathologist must examine. Routine histology with H&E staining will identify micrometastatic disease in an estimated 15% to 20% of SLN dissections; more sections and immunohistochemical (IHC) staining will identify an estimated 8% to 10% additional patients with stage III melanoma.
In the AJCC melanoma database,2 the 5-year survival rate of patients with primary tumors greater than or equal to 1.0 mm was 65% when nodes were staged negative with clinical exam, 75% when nodes removed during elective lymph node dissection (ELND) were negative, and 90% when patients were staged to be node negative with the SLN procedure.5 These differences underscore the importance of accurate assessment of the regional lymph nodes for occult metastases.
Our first assay for occult metastases was a simple tissue culture method.6,7 Malignant cells grew from 25% of the lymph nodes that were initially assessed as negative by the pathologist.8 Patients whose lymph nodes were histologically negative but tissue culture positive had a higher recurrence rate and lower survival rate than patients whose SLNs were negative by both assessments. Because the tissue culture assay was too cumbersome and inefficient for widespread use, Lakeland Regional Cancer Center (LRCC) investigators subsequently developed a more reproducible, highly sensitive method to detect micrometastases by examining SLNs for the presence of tyrosinase messenger RNA (mRNA), a material found in melanocytes. Lymph nodes from 29 patients with intermediate-thickness melanoma were analyzed by standard pathologic staining and reverse transcriptase polymerase chain reaction (RT-PCR).9 Eleven samples (38%) were pathologically positive; 19 (66%) were RT-PCR positive, including all of the pathologically positive samples. From this study it was concluded that the RT-PCR method is an extremely sensitive, specific, reproducible and efficient technique for the identification of micrometastases in patients with melanoma. Whereas microscopic evaluation can identify one melanoma cell in up to 104 normal lymphocytes and S-100 and HMB-45 immunostains can detect one abnormal cell in up to 105 lymphocytes,10 molecular biology assays can identify one abnormal melanoma cell in 106 normal lymphocytes.9
METHODS
Patient Population
Between 1992 and 2002, patients were recruited from the Cutaneous Oncology Program at the H. Lee Moffitt Cancer Center (MCC) at the University of South Florida in Tampa and the LRCC, in Lakeland, Florida. Appropriate institutional review board approval was obtained. Each patient consented to participate voluntarily. All patients met the following inclusion criteria: (1) diagnosis of cutaneous melanoma; (2) melanoma with Breslow thickness greater than 0.75 mm, unless other high-risk factors for metastasis existed; (3) no grossly palpable disease present on physical examination; (4) no sign or symptoms of local, regional, or systemic metastatic disease; and (5) no multiple primary melanomas. These criteria ensured that all patients enrolled in the study had clinical stage I or II melanoma. Patients whose SLN biopsy, tissue culture, or PCR test (negative for the ß-actin control) was unsuccessful were removed from the study.
Preoperative Lymphoscintigraphy and Intraoperative Lymphatic Mapping
All patients underwent preoperative lymphoscintigraphy to determine the actual lymphatic basin(s) at risk for metastases and the approximate number and location of the SLNs in the basin. In brief, technetium-99 sulfur colloid, filtered with a .2-micron filter (Syncor, Tampa, FL) was injected around the primary tumor or the previous biopsy site. Dynamic and delayed images were obtained to show the lymphatic drainage pattern of the primary tumor, as well as the anatomic relationships of potential SLNs. An intradermal tattoo was used to mark the location of potential SLNs. This procedure has been described in detail elsewhere.11
Patients were taken to the operating room for intraoperative lymphatic mapping and SLN biopsy under general anesthesia. The lymphatic mapping technique used both a vital blue dye (Lymphazurin, USSC, Norwalk, CT) and a radiocolloid to identify the SLN intraoperatively. Because the SLN was harvested 2 to 24 hours after lymphoscintigraphy, radioactivity in SLNs was still detectable and reinjection of radiocolloid was avoided. Vital blue dye was injected around the primary tumor or previous biopsy site at the beginning of the surgical procedure. Ten to 15 minutes after the injection of blue dye, a small incision was made at the site of the "hot spot" in the regional basins identified with the hand-held probe. Lymph nodes with a blue-stained afferent lymphatic, containing blue dye, or with appropriate levels of radioactivity (SLN activity vs. a neighboring non-SLN activity >10:1 ex vivo) detected by a hand-held gamma probe were harvested as SLNs. Subsequently, patients underwent a 1.0-cm wide local excision (WLE) for melanomas less than 1.0 mm in thickness or a 2.0-cm WLE for all other tumor thicknesses.
SLN Examination
SLNs were bisected. Half of each SLN was processed for routine pathology examination: the specimen was sectioned at 2 to 3–mm intervals and embedded in paraffin. Each block was sectioned at one to three levels, depending on the size of the tissue in the block, and stained with H&E. If no melanoma cells were found with H&E staining, IHC staining with S-100 antibody was performed by means of an avidin-biotin complex immunoperoxidase technique with diaminobenzidine chromogen. If specimens were negative by H&E but positive by IHC, the H&E slides were carefully reviewed again, or more sections were cut and stained with H&E to verify the presence of metastatic melanoma cells by examining cytologic features. Only samples that were confirmed positive by H&E staining were reported as positive. If the metastatic cells were first identified with the S-100 stain, then the pathologist used the H&E stain to evaluate the cytology of S-100-positive cells. The histologic examination of the SLN has been described in detail elsewhere.6
The second half of each SLN was submitted for tissue culture or RT-PCR examination. Specimens submitted for tissue culture were mechanically dispersed into a single-cell suspension and placed in standard tissue culture medium (Dulbecco’s modified Eagle’s medium). Culture plates were fed and then inspected at 2 and 4 weeks for malignant cell growth. Cell populations were identified by staining with IHC for melanoma-specific markers.
SLN specimens submitted for RT-PCR examination were processed for RNA extraction and RT-PCR, as previously described.11 Primers used also have been described previously.12 In brief, samples were sent immediately from the operating room to the laboratory, where they were trimmed of external fat and rapidly frozen at -80°C. Total RNA was extracted by a phenol-guanidinium thiocyanate method. A cDNA library was constructed by using oligo-dT as the primer for reverse transcription. A separate PCR assay for the mRNA of the ß-actin housekeeping gene was performed to verify general mRNA integrity. A nested PCR for the detection of tyrosinase cDNA was conducted on ß-actin-positive samples to determine lymph node metastatic status. The first round of PCR (30 cycles) generates a 284–base pair (bp) DNA fragment, and the second round (30 cycles) with nested primers generates a 207-bp product. The second-round PCR products were analyzed by electrophoresis in a 2% agarose gel (BioRad, CA) and stained with ethidium bromide. If an SLN sample produced the 207-bp fragment, it was considered PCR-positive.
Adjuvant Therapy and Follow-Up Schedule
Patients who had tumor-positive nodes by routine histopathology (H&E or S-100 staining) were offered complete lymph node dissection and/or interferon 
-2 therapy.13 Tissue culture or RT-PCR results were not used for clinical decision-making, and all patients who were negative by routine histology were observed.
Patients were followed at least every 3 to 6 months for the first and second years postsurgery and yearly afterwards. Patients were checked carefully by physical examination, including radiography or other diagnostic tools, when indicated. Recurrence was confirmed by physical examination and documented in the melanoma database.
Statistics
Survival functions were generated for relapse-free survival with the product-limited method of Kaplan-Meier. Overall survival was calculated from the date of diagnosis to the date of death. Relapse-free survival was calculated from the date of diagnosis to the date of first recurrence. Patients not experiencing these events were considered censored at the date of last contact. The distribution of each variable was evaluated, and measures of central tendency and variance were estimated. Univariate and multivariate regression analyses were performed, with prognostic variables based on the primary tumor (tumor thickness, Clark level, ulceration, and location), as well as clinical variables (age, sex) and the histology and RT-PCR status of the SLN. Chi-square statistics was used for comparing differences among different categories, which were formed according to PCR and histology status. An level of .05 and 95% confidence intervals were used throughout the analysis.
RESULTS
Of the 326 patients who underwent SLN biopsy between 1992 and 2002, 69% were males. The mean age of the entire group was 57 years. The mean thickness of the primary cutaneous melanoma was 2.18 mm. Most patients (94%) had Clark level III and IV melanomas. Ulceration was present in 64% of the specimens. The mean follow-up of the group was 37 months. Two separate dermatopathologists read each H&E and IHC slide. In routine examination under H&E staining, 64 patients (19%) had metastatic melanoma in one or more SLNs. In 16 of these 64 patients (25% of the patients with metastatic disease discovered with routine methods, or 4.8% of the total population), the metastatic cells were initially found with S-100 IHC staining and confirmed to be malignant with H&E stain. This was after metastatic cells had not been detected by the initial H&E screening. Reexamination, and in some cases further sectioning, verified the presence of metastatic melanoma cells in all of these patients. These cases would have been inaccurately staged with ELND specimens.
Results of RT-PCR assay of the SLN have been correlated with clinical outcome.14 Patients whose SLNs were histologically positive and PCR positive had a recurrence rate of 42% at 3 years, whereas patients whose SLNs were negative in both assays had a recurrence rate at 3 years of 6.6%. The interesting group is patients whose SLNs are histologically negative but PCR positive. These patients have an intermediate prognosis, with 22% recurring at 3 years. In addition, RT-PCR positivity of the regional nodes or SLNs correlates with tumor thickness at diagnosis, a prognostic factor that is linearly related to survival. Patients with relatively thin melanomas (.76 to 1.5 mm) have an RT-PCR-positive-node rate of 33%, whereas 80% of the individuals with thick melanomas have tyrosinase mRNA in the regional node.15
DISCUSSION
The Florida Melanoma Trial (FMT) (Fig. 1) involves 10 institutions and will enroll over 3200 patients with melanoma over a 5-year period. It will determine whether a complete node dissection (CLND) after a positive SLN biopsy contributes to disease control and survival. In previous trials16,17 only 15% to 20% of the patients had positive nodes, and thus the majority could not benefit from a CLND. In the FMT, only those patients with confirmed evidence of metastatic disease in the regional basin (positive SLN) are eligible to be randomized to CLND plus adjuvant therapy with interferon or to interferon alone (part II of the FMT).
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The Eastern Cooperative Oncology Group (ECOG) has performed three studies to assess interferon -2b (IFN) therapy for stage III melanoma. ECOG 168413 has shown prolonged disease-free survival (DFS) and overall survival (OS) after 1 year of high-dose IFN. ECOG 1690,18 in a larger number of patients, showed a significant increase in DFS after high-dose IFN versus low-dose IFN or no treatment, but the OS advantage was not apparent. The lack of a survival advantage in this trial was attributed in part to effective salvage therapy with high-dose IFN in those patients who recurred in the low-dose or no-treatment arm. ECOG 169419 showed a significant DFS and OS advantage for high-dose IFN versus a ganglioside vaccine. Because high-dose interferon is the only approved postoperative adjuvant therapy for stage III melanoma, all patients randomized in part II of the FMT will receive systemic high-dose IFN.
Use of RT-PCR to detect tyrosinase mRNA has been applied in many studies as a marker for melanoma cells.9,20–24 Lymph nodes that are histologically positive for melanoma are RT-PCR positive in 94% to 100% of cases.9,15,22 What accounts for the larger percentage of patients whose SLNs are histology negative but RT-PCR positive? Sampling error could play a role, but all these SLNs are grossly negative when they are bivalved; thus, a sampling error should apply equally to both halves. More than likely, the RT-PCR assay is detecting missed micrometastatic disease that is clinically relevant. This is reflected in the fact that patients whose SLN is negative in routine histology but positive in RT-PCR have a significantly increased recurrence rate and decreased survival.14 Furthermore, RT-PCR and IHC measure products from different genes. Investigators have argued that these molecular biology assays may be detecting low-volume disease that the immune system would ordinarily destroy or that the RT-PCR assay is detecting malignant cell byproducts instead of viable metastatic cells. Again, the worse prognosis associated with RT-PCR upstaging argues against this hypothesis.
Five percent of the SLNs contain benign nevus cell rests. Nevus cells are positive for tyrosinase mRNA and would be a source of false-positive RT-PCR results. These nevus cell rests are usually easily distinguished from malignant cells in the lymph node on the basis of their cytologically benign appearance and their usual location in the fibrous capsule or trabeculae of the lymph node. If a benign nevus cell rest is found on routine histology, then the SLN specimen contains a possible source of false-positive RT-PCR results. The FMT will employ multiple markers (tyrosinase and MART-125) and use a quantitative RT-PCR assay in an attempt to eliminate false-positives caused by nevus cell rests in the SLN.
RT-PCR assays for metastatic melanoma in the SLN have been developed by other institutions with slight variation from the LRCC protocol. Most are based on the tyrosinase probe, but others have combined tyrosinase with other melanoma-specific markers in a multiple marker assay. Universally, their reports20–24 have shown that the RT-PCR assay will identify a population of patients with missed micrometastatic disease in their SLNs. Most also showed that the upstaged population has a higher recurrence rate and a lower survival rate than melanoma patients whose SLN is RT-PCR negative.
These single-institution studies are the basis for the Sunbelt Melanoma Trial, a prospective national trial with 65 participating institutions. Patients with melanomas greater than 1.0 mm in thickness undergo preoperative lymphoscintigraphy to define all the basins at risk for metastases and then intraoperative lymphatic mapping and SLN biopsy. If the SLN is histologically negative, a multiple-marker RT-PCR assay is performed; a positive result is based on expression of tyrosinase and at least one other marker. This alleviates the problem of benign nevus cell rests in 5% of the SLNs, which would be positive with the tyrosinase RT-PCR. The Sunbelt Melanoma Trial attempts to confirm in a multicenter setting the clinical relevance of missed micrometastatic disease and the optimal treatment in this setting. The FMT will ultimately be used to determine the role of CLND in patients with melanoma and will be the first trial to incorporate molecular staging in treatment decisions.
FOOTNOTES
In the future, melanoma may be "ultrastaged" by examining the sentinel lymph nodes, peripheral blood, and bone marrow, thus improving identification of patients who are surgically cured of their disease. The lymphatic mapping procedure is the most accurate way to determine the tumor status of the regional lymph nodes.
Received for publication December 17, 2003. Accepted for publication December 29, 2003.
REFERENCES
This article has been cited by other articles:
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  A. Govindarajan, D. M. Ghazarian, D. R. McCready, and W. L. Leong Histological Features of Melanoma Sentinel Lymph Node Metastases Associated with Status of the Completion Lymphadenectomy and Rate of Subsequent Relapse Ann. Surg. Oncol., February 1, 2007; 14(2): 906 - 912. [Abstract] [Full Text] [PDF]
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 D. R. Berk, D. L. Johnson, A. Uzieblo, M. Kiernan, and S. M. Swetter Sentinel Lymph Node Biopsy for Cutaneous Melanoma: The Stanford Experience, 1997-2004 Arch Dermatol, August 1, 2005; 141(8): 1016 - 1022. [Abstract] [Full Text] [PDF]
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