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Originally published as JCO Early Release 10.1200/JCO.2003.07.982 on August 11 2003 © 2003 American Society for Clinical Oncology
Advances in Molecular Staging of Melanoma Patients: Multimarker Analysis of Archival Lymph Node TissueDepartment of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY THE PRESENCE of regional lymph node metastasis is an important predictor for clinical outcome of patients with primary cutaneous melanoma. Once nodal involvement by melanoma is detected, the 5-year survival of patients is reduced by approximately 40% compared with those who have negative nodes, and the characteristics of the primary tumor, such as its thickness, become less important for patient survival.1 Accordingly, much attention has focused on optimizing the detection of metastatic disease in regional lymph nodes. The technique of sentinel lymph node (SLN) biopsy has proven highly accurate for predicting the pathologic status of regional lymph nodes,2 and has become the standard of care for patients whose primary cutaneous melanoma measures at least 1 mm in greatest thickness.3,4 Being able to focus on one or only a few lymph nodes has allowed pathologists to search more comprehensively for metastatic tumor deposits by examining multiple tissue sections and by using immunohistochemical techniques that facilitate the recognition of small tumor deposits by light microscopy. However, conventional histologic analysis, even if enhanced by multiple sections and immunostains, has an inherent limitation: routine histologic processing protocols assess approximately only 1% to 5% of the lymph node tissue for examination. Unless a node is step-sectioned in its entirety, which would be impractical, there is always the risk of histologic sampling error. Searches for more sensitive methods of assessing the status of the SLN have led to the application of molecular techniques, which allow for analysis of the entire nodal tissue not used for routine histology.5 Instead of looking for melanoma cells under the microscope, transcripts of target genes, such as tyrosinase or other melanocyte differentiation markers, are amplified by reverse transcriptase–polymerase chain reaction (RT-PCR).6–10 The detection of tyrosinase gene transcripts in SLNs of patients with melanoma is much more sensitive than the recognition of melanoma cells by light microscopy.5–10 In some studies, up to 65% of patients were upstaged by such molecular data.6 However, there is a potential pitfall in the assumption that tyrosinase gene expression confers the same prognostic information as pathologically documented metastatic disease. Benign melanocytes can be present in lymph nodes as nodal nevi, with reported incidences varying from less than 5% to as high as 22% of lymphadenectomy specimens in melanoma patients,11 and may account for a positive RT-PCR result for tyrosinase. Furthermore, illegitimate transcription or technical artifacts may yield false-positive results. Therefore, correlation of molecular results with histology and patient outcome is critical to judge the clinical significance of molecular results.6 Preliminary data have suggested that molecular staging may be able to identify patients with a histologically negative SLN who are at risk for recurrence.5 However, clinical follow-up is limited (average of 28 months) and evidence is emerging that the prognostic value of tyrosinase gene expression as currently measured by RT-PCR decreases with longer follow-up of the patients (D. Coit, personal communication, April 2003). Although most prior attempts of molecular staging of melanoma patients have relied on the detection of a single marker (usually tyrosinase) in frozen tissue, several investigators are taking the field of molecular staging into new directions. First, instead of examining a single marker, multiple markers are being analyzed to enhance sensitivity and specificity of the assay. Another novelty is the application of RT-PCR analysis to formalin-fixed and paraffin-embedded archival tissue.9,10 This is an exciting new development. The use of archival material has great appeal for clinical care and investigational studies. It simplifies the handling and storage of tissue that can be examined first by standard histologic and immunohistochemical techniques before the remaining material is used for molecular analysis. It greatly facilitates the design of research studies by allowing inclusion of patients whose lymph node tissue was not frozen and by providing access to investigate molecular staging in large groups of patients whose clinical courses are fully characterized. This new approach of multimarker analysis on archival material is exemplified by the work of Kuo et al10 in this issue of the Journal of Clinical Oncology. The authors studied the expression of four melanocyte differentiation antigens, including tyrosinase, melanoma antigen recognized by T cells-1 (MART-1), tyrosinase-related protein-1 (TRP-1), and TRP-2. The results are impressive. The sensitivity of RT-PCR analysis of archival material was high: at least one marker could be detected in all nodes containing metastatic melanoma. Twenty-five percent of patients with histologically negative SLNs were upstaged using two or more markers. Of these, 80% developed a recurrence. Patients with histologically negative SLNs and zero or one expressed marker had a significant improved disease-free and overall survival compared with those with two or more markers (median follow-up, 55 months). Obviously, such promising results need to be confirmed, given the interlaboratory variation in the conditions and detection methods for RT-PCR studies. One particular aspect of the study is intriguing and difficult to explain. Why does the number of expressed markers make such a difference in predicting clinical outcome? All four markers tested in the study encode melanosome-associated proteins. Three are directly involved in melanin pigment synthesis. It would have been easier to understand the impact of one additional marker, if it were involved in a different cellular function. Another concern is the possibility of false-positive results. Because Bieligk et al6 found a false-positive RT-PCR result in 11% of their lymph nodes, it is surprising that Kuo et al10 did not encounter a single false-positive test result in 25 control lesions, especially in light of the fact that multiple markers were used. Although they refer to these markers as melanoma markers, they are melanocyte differentiation antigens, which cannot distinguish between benign melanocytes and melanoma cells. Thus, a small nodal nevus, unless it is morphologically detected in the sections taken for routine histology or immunohistochemistry, may still account for a false-positive test results with the panel of differentiation markers chosen by the authors. The inclusion of additional markers of different biologic significance, such as cancer-associated genes, merits further investigation in this regard to enhance the specificity of RT-PCR analysis. As the work of Kuo et al10 illustrates, the field of molecular staging is progressing into new and promising directions. Multiple markers can successfully be detected by RT-PCR in archival material, and preliminary data suggest a potential prognostic role for this approach. However, additional studies are needed to validate the results and to determine their clinical significance. AUTHOR’S DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author indicated no potential conflicts of interest. REFERENCES
1. Ferrone CR, Panageas KS, Busam K, et al: Multivariate prognostic model for patients with thick cutaneous melanoma: Importance of sentinel lymph node status. Ann Surg Oncol 9:637–645, 2002
2. Morton DL, Wen D, Wong JH, et al: Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 127:392–399, 1992
3. Balch CM, Buzaid AC, Soong SJ, et al: Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma. J Clin Oncol 19:3635–3648, 2001
4. McMasters KM, Reintgen DS, Ross MI, et al: Sentinel lymph node biopsy for melanoma: Controversy despite widespread agreement. J Clin Oncol 19:2851–2855, 2001
5. Shivers SC, Wang X, Li W, et al: Molecular staging of malignant melanoma: Correlation with clinical outcome. JAMA 280:1410–1415, 1998 6. Bieligk SC, Ghossein R, Bhattacharya S, et al: Detection of tyrosinase mRNA by reverse transcriptase polymerase chain reaction in melanoma sentinel nodes. Ann Surg Oncol 6:232–240, 1999[Abstract]
7. Ribuffo D, Gradilone A, Vonella M, et al: Prognostic significance of reverse transcriptase-polymerase chain reaction-negative sentinel nodes in malignant melanoma. Ann Surg Oncol 10:396–402, 2003
8. McMasters KM: Molecular staging of melanoma: Sensitivity, specificity, and the search for clinical significance. Ann Surg Oncol 10:336–337, 2003 9. Bonin S, Niccolini B, Calacione R, et al: Molecular analysis of sentinel lymph nodes: An open question. J Eur Acad Dermatol Venereol 16:34–39, 2002[CrossRef][Medline]
10. Kuo CT, Hoon DSB, Takeuchi H, et al: Prediction of disease outcome in melanoma patients by molecular analysis of paraffin-embedded sentinel lymph nodes. J Clin Oncol 21:3566–3572, 2003 11. Carson KF, Wen DR, Li PX, et al: Nodal nevi and cutaneous melanomas. Am J Surg Pathol 20:834–840, 1996[CrossRef][Medline]
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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