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Journal of Clinical Oncology, Vol 24, No 18 (June 20), 2006: pp. 2849-2857 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.03.2342 Prospective Multi-Institutional Study of Reverse Transcriptase Polymerase Chain Reaction for Molecular Staging of Melanoma
From the Division of Surgical Oncology, Department of Surgery, University of Louisville, James Graham Brown Cancer Center and Center for Advanced Surgical Technologies (CAST); Advertek Inc, Louisville, KY; Department of Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston; Department of Surgery, Dallas Surgical Group, Dallas, TX; Lakeland Regional Cancer Center, Lakeland, FL; Department of Surgery, LDS Hospital, Salt Lake City, UT; Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ; Department of Surgery, University of Alabama, Birmingham, AL; Melanoma Unit of the Yale Cancer Center, Department of Surgery, Yale University School of Medicine, New Haven, CT; Albany Surgical PC, Albany, GA; Department of Surgery, University of Cincinnati, Cincinnati, OH; Department of Surgery, University of Arkansas for Medical Sciences, Little Rock, AK; and the National Genetics Institute, Los Angeles, CA Address reprint requests to Kelly M. McMasters, MD, PhD, Department of Surgery, 315 E Broadway, Room 305, University of Louisville, Louisville, KY 40292; e-mail: mcmasters{at}louisville.edu
PURPOSE: To evaluate the prognostic significance of molecular staging using reverse transcriptase polymerase chain reaction (RT-PCR) in detecting occult melanoma cells in sentinel lymph nodes (SLNs) and circulating bloodstream.
PATIENTS AND METHODS: In this multicenter study, eligibility criteria included patient age 18 to 71 years, invasive melanoma RESULTS: A total of 1,446 patients with histologically negative SLNs underwent RT-PCR analysis. At a median follow-up of 30 months, there was no difference in DFS, DDFS, or OS between the RT-PCR–positive (n = 620) and RT-PCR–negative (n = 826) patients. Analysis of PBMC from 820 patients revealed significant differences in DFS and DDFS, but not OS, for patients with detection of more than one RT-PCR marker in peripheral blood. CONCLUSION: In this large, prospective, multi-institutional study, RT-PCR analysis on SLNs and PBMCs provides no additional prognostic information beyond standard histopathologic analysis of SLNs. Detection of more than one marker in PBMC is associated with a worse prognosis. RT-PCR remains investigational and should not be used to direct adjuvant therapy at this time.
The most important predictor of survival for patients with early-stage melanoma is the regional nodal status.1 Modern management of melanoma includes sentinel lymph node (SLN) biopsy. The SLN is the first node that receives lymphatic drainage from the primary tumor; therefore, it reflects the status of the entire basin.2-5 SLN biopsy represents a minimally invasive method of nodal staging. Despite the accuracy of SLN biopsy in documenting nodal status, a significant number of SLN-negative patients (approximately 10% to 20%) will develop metastatic disease.6-9 Attempts to identify the stage I and II patients at greatest risk for recurrence have focused mainly on detection of melanoma-specific mRNA using reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is a highly sensitive technique that can detect a single melanoma cell within 1 million normal cells.10,11 RT-PCR for metastatic melanoma has been studied in various tissue types, including SLNs12-15 and blood.12,16,17 The tyrosinase gene has been most commonly evaluated by RT-PCR in melanoma research.12-15,18-21 Other markers, such as MART1,13,14,18,20,22 MAGE3,14,18 and others,14,20 have been studied. Based on these data, we sought to evaluate the prognostic significance of RT-PCR of SLNs and peripheral-blood mononuclear cells (PBMCs) to detect melanoma patients at greatest risk of recurrence and mortality.
The Sunbelt Melanoma Trial The Sunbelt Melanoma Trial is a prospective, randomized trial involving 79 institutions that is evaluating the role of RT-PCR for ultrastaging, lymphadenectomy, and adjuvant interferon alfa-2b for patients with early nodal metastasis.23 This study was approved by the institutional review board of each institution. Patients age 18 to 71 years with invasive melanoma  1 mm Breslow thickness and without clinical evidence of regional or distant metastasis were eligible. Random assignment was accomplished using a randomized permuted block with stratification by tumor thickness and ulceration, with reassignment to ensure that each center was roughly balanced as to the number of patients in each arm. Following Zelen,24 reassignment was done if the difference in treatment group sample sizes was more than a small randomly chosen positive integer. Power and sample size calculations were based on a comparison of survival time between the treatment arms with the following assumptions: 5-year accrual time, 10-year minimum follow-up time (for therapeutic results), right random censoring, one-sided significance level of .05, and a power level of at least 80% for detecting a 10% change in overall survival.
After informed consent was provided, patients underwent excision of the primary melanoma and SLN biopsy using intradermal injection of technetium sulfur colloid around the primary tumor site. A lymphoscintigram was obtained and a hand-held gamma probe was used intraoperatively to guide SLN identification. Intradermal injection of isosulfan blue dye (1 to 5 mL) was performed in the majority of patients as well. All blue nodes and all nodes A portion of each SLN (defined as one fourth of the lymph node or a 2-mm3 portion of the node, whichever was smaller) was snap-frozen on dry ice or liquid nitrogen and stored at –80°C until it was shipped on dry ice. If more than one SLN was found, each SLN was processed identically. The remaining SLN tissue was processed by hematoxylin and eosin (H and E) staining at multiple levels, with at least five sections per block, along with two additional random sections for S-100 immunohistochemistry (IHC). A histologically positive SLN was defined as evidence of metastatic tumor cells identified by either H and E or IHC. A central pathology review committee evaluated the first 10 patients from each participating institution, as well as all samples of SLNs containing metastases. Patients also had blood drawn at the time of consent, 3 months postoperatively, and annually thereafter. As a result of logistic issues related to processing blood specimens, several sites were granted exemptions from drawing the blood samples, resulting in fewer blood samples for RT-PCR testing. The first tube of blood drawn was discarded to diminish the chance of contamination from the skin plug generated during venipuncture. After collection of 5 mL of blood in EDTA-containing tubes, peripheral blood mononuclear cells (PBMCs) were isolated with a hypotonic density gradient solution (DOT kit, National Genetics Institute, Los Angeles, CA) as described previously,26 and total RNA was shipped to the central laboratory on dry ice. SLNs and PBMCs were processed and analyzed by a central laboratory (National Genetics Institute) that was blinded to clinical and pathologic data. Total RNA was extracted using TRIREAGENT (Molecular Research Center Inc, Cincinnati, OH), quantified by absorbance at 260 nm, then precipitated with ethanol.27 The quality of the RNA isolated was demonstrated by amplification and detection alongside beta-actin. RT-PCR was performed with specific primers for tyrosinase, MART1, MAGE3, and GP-100, followed by Southern blot detection.26-28 The Southern blot signals were analyzed by determining optical band density for samples and controls. Test samples with optical densities more than 50% that of the negative controls were considered positive. Negative controls for SLNs included RNA from negative nodes as well as RNA from human melanoma cell lines. PBMC were collected from healthy controls and RNA from these negative controls was tested side by side with patient samples. The a priori definition of a positive SLN RT-PCR test was detection of tyrosinase mRNA plus at least one other marker. Using this definition, we had no false-positive results when 100 nodes from patients without melanoma were analyzed during initial assay validation studies.29 RT-PCR for PBMCs was considered positive if any marker was detected at any point in time. The study population for RT-PCR testing of SLNs included patients who were histologically negative after SLN analysis. The PBMC RT-PCR population, however, included patients with both histologically negative and positive SLNs.
Disease-free survival (DFS) was calculated from the date of random assignment to the date of the first recurrence. Distant–disease-free survival (DDFS) was calculated from the date of random assignment until the date of first distant recurrence. Overall survival (OS) was calculated from the date of random assignment to the date of death. Survival distributions were estimated using Kaplan-Meier methods and the log-rank test was used to assess the statistical significance of differences in DFS, DDFS, and OS between groups. Because of relatively early follow-up, DFS and DDFS might be surrogates for changes in OS, or reflect differences in the pattern of recurrence. Therefore, all three measures were evaluated. For continuous variables, P values are for two-sample, equal variance t tests. For categoric variables, P values represent
RT-PCR Analysis of SLNs The Sunbelt Melanoma Trial was open for accrual from June 1997 through October 2003. The data analysis for this study includes follow-up data through April 2005. The median follow-up for this cohort was 30 months. The clinical and pathologic features of the patient population are listed in Tables 1 and 2. For this cohort, RT-PCR analysis was performed in 3,505 SLNs from 1,446 H and E/IHC-negative patients. Among patients with RT-PCR–positive SLN, the median of RT-PCR-positive SLN was 2 (range, 1 to 10). Table 3 lists this information and the SLN RT-PCR results for individual markers.
There were no differences in DFS (P = .61; Fig 1A), DDFS (P = .093; Fig 1B), or OS (P = .36; Fig 1C) between RT-PCR–negative and RT-PCR–positive patients. Similarly, there were no differences in DFS (P = .58; Fig 2A), DDFS (P = .052; Fig 2B), or OS (P = .15; Fig 2C) when analyzed by the number of markers expressed. In total, 63 (10.2%) SLN RT-PCR–positive patients and 91 (11.0%) SLN RT-PCR–negative patients have developed recurrent disease (P = .60). There were no differences in the patterns of recurrence (local, regional, distant) between PCR-negative and PCR-positive patients (for either SLN or PBMC).
Furthermore, using P = .05, there were no statistically significant differences in DFS, DDFS, or OS between patients with RT-PCR–positive or –negative SLN when analyzed by treatment received (observation, lymph node dissection, or lymph node dissection plus adjuvant interferon alfa-2b [data not shown]). In addition, there was no difference in DFS (P = .57), DDFS (P = .06), or OS (P = .47) for patients with multiple RT-PCR–positive SLNs when compared with those with 0 or 1 RT-PCR–positive SLN. For SLNs, all prognostic factors in Table 1 were included in multivariate Cox proportional hazards models. After simultaneous adjustment for these known prognostic factors, RT-PCR (DFS, P = .23; DDFS, P = .14; OS, P = .41) was not significant in any of the Cox models. For blood RT-PCR (Table 2), similar models were considered. RT-PCR for PBMCs was likewise not significant for any survival end points (DFS, P = .89; DDFS, P = .10; OS, P = .07). A variable measuring the number of days from the first blood sample until a patient tested positive for blood RT-PCR or the last blood sample was drawn was added to the Cox models and found to be significant, but testing positive for RT-PCR still did not negatively influence any of the survival end points. Additional analysis was performed to determine, what if any, effect the number of positive markers had. For SLNs, there was no effect of positive marker count on DFS (P = .7) or OS (P = .2) even when considering factors known to influence outcome, such as sex, tumor site, thickness, and ulceration. However, there was a slight effect for males (P = .022) on DDFS. For the PBMC cohort, there were only 16 patients with more than one positive marker, making similar analyses of these data not useful. The associations between characteristics in Tables 1 and 2 and the survival measures were investigated. It was found that for RT-PCR–negative patients, but not RT-PCR–positive patients, males generally had worse survival, and those males and females with head-neck melanomas had worse survival. As would be expected, ulceration adversely influenced all survival measures for both RT-PCR–positive and –negative patients. Clark level influenced survival minimally.
RT-PCR Analysis of PBMCs
SLN status is the strongest predictor of survival for early-stage melanoma.9,30-33 The burden of disease within the node appears to affect outcome as well,34 thus providing additional impetus to identify patients with micrometastatic disease who may be candidates for additional therapy. In our study, which is the largest study of molecular staging of melanoma to be reported, we found no clinically significant benefit for RT-PCR analysis. Others have observed similar results,21 and concluded that RT-PCR does not give prognostic information above that provided by thorough SLN histopathology. However, other smaller studies have demonstrated varying degrees of predictive value for RT-PCR testing of SLNs.12,18-20,22,35-37 Shivers et al35 found that PCR for tyrosinase predicted recurrence and overall survival. Interestingly, there was only one recurrence of 44 (2%) histologically negative, PCR-negative SLN patients and six of 47 (13%) recurrences in the patients whose SLNs were histologically negative and PCR positive.35 Li et al38 have shown that detection of tyrosinase mRNA in SLNs correlates with histological predictors of adverse outcome. In addition, in a study of 129 patients, Gradilone et al36 demonstrated that expression of PCR markers tyrosinase and/or MIA strongly correlates with DFS. The specificity of tyrosinase has been questioned, mainly because of the possibility of a false-positive result.39 This has led investigators to evaluate multiple-marker RT-PCR to improve specificity.14,18,20 Using a multiple-marker RT-PCR assay (MART1, MAGE3, GalNAc-Tß, and Pax3), Takeuchi et al40 found prognostic value for SLN RT-PCR. In a much smaller, retrospective study, Bostick et al18 were able to correlate multiple-marker PCR (tyrosinase, MART1, MAGE3), and histological evidence of SLN metastasis with increased recurrence. Others have reported similar results in small studies.15,22,38 Despite a multimarker RT-PCR assay, sensitive Southern blot detection, and analysis at a central laboratory, we were not able to demonstrate prognostic significance for RT-PCR. Experimental and procedural variables that may affect RT-PCR include specimen processing, RNA condition, choice of primers, and RT-PCR conditions. Because the study began in 1997, the assay was not based on semiquantitative real-time RT-PCR, which would be the present standard. Perhaps a more quantitative assessment of RT-PCR markers would result in a more predictive test. The fact that the assay had no false-positive results in 100 nonmelanoma patient lymph nodes suggests that indiscriminate amplification of nonspecific mRNA was not likely the main problem.29 mRNA was detected, but low levels of expression may be clinically insignificant. It is possible that a small number of metastatic melanoma cells within a node do not establish themselves as clinically significant metastases, and/or that the patient’s own defenses eliminate these cells. In addition, the fact that 43% of patients with H and E/IHC-negative SLNs had a positive RT-PCR test, which greatly overestimates the fraction of patients likely to experience recurrence, suggests that greater assay specificity is needed. Perhaps other markers would have provided different results; however, 75% of the 647 histologically positive SLNs were positive for tyrosinase. This percentage is similar to previous reports.18,21,41,42
Another consideration is the fact that 11% of the H and E/IHC- and PCR-negative patients have experienced recurrence. This might be explained by sampling error, given that Another potential method of molecular staging of melanoma is detection of metastatic melanoma in blood. In the early 1990s, Smith et al16 demonstrated the feasibility of RT-PCR for melanoma in peripheral blood. Subsequently, several studies have suggested that RT-PCR evidence of circulating melanoma cells may have prognostic significance, although the results are somewhat mixed.12,17,28,43-48 In this study, RT-PCR analysis of PBMCs demonstrated that the prognosis of patients with one marker was no different than for those who had no markers detected. Expression of more than one marker was associated with worse DFS and DDFS; OS was not different. However, only 2% of patients had evidence of more than one PBMC RT-PCR marker, which underestimates the fraction of patients that will experience recurrence. Thus, the PBMC RT-PCR assay, using more than one marker detected as the definition of a positive test, is not likely to have sufficient sensitivity to make it clinically useful. Because the study did not randomly assign patients based on PBMC RT-PCR results, the protocol did not include an a priori definition of a positive PBMC RT-PCR result: the PBMC results were observational. The goal of the PBMC studies was to provide a definition of a positive test based on analysis of these data. Five milliliters of blood may not have been sufficient to capture a limited number of circulating tumor cells, although this volume is similar to those used by other investigators.46-49 Finally, although individual sites processed the blood samples, a central laboratory performed the PCR testing. The central laboratory provided the processing kits, which included a simple, step-by-step procedure designed to reduce variability, and although significant variability is unlikely, it is possible. Another caveat is the relatively short median follow-up for this study (30 months). Kammula et al21 found initial differences in survival for patients with RT-PCR–positive versus –negative SLNs that disappeared with longer follow-up, and recommended that such studies should include long-term follow-up. In our study, we found the same phenomenon: initial results with limited follow-up (12 months) demonstrated divergence of the disease-free survival curves, which subsequently converged. Given the size and statistical power of our study, it is unlikely that additional follow-up will result in a significant divergence of the curves. In conclusion, using a large, multicenter, randomized, prospective study, we did not find any predictive value of SLN RT-PCR analysis. Detection of more than one marker in PBMC was associated with worse prognosis, although the clinical utility of this assay is likely limited. Additional refinements in molecular staging of melanoma may hold promise for the future. However, we were not able to demonstrate that molecular staging of melanoma would provide significant prognostic information above and beyond standard SLN histopathology. At present, RT-PCR for molecular staging of melanoma should be considered investigational, and should not be used to direct therapy.
Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000-99,999 (C)
We thank the Sunbelt Melanoma Trial coordinators, the Sunbelt Melanoma Trial Study Group, and the Pathological Review Committee for their dedication and participation.
Supported by a grant from Schering Oncology-Biotech and the Center for Advanced Surgical Technologies-Norton Hospital, Louisville, KY. Schering Oncology-Biotech had no access to data, no input into the analysis of the data, and has not participated in the preparation or review of the manuscript. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Pantel K, Cote RJ, Fodstad O: Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst 91:1113-1124, 1999 44. Hoon DS, Bostick P, Kuo C, et al: Molecular markers in blood as surrogate prognostic indicators of melanoma recurrence. Cancer Res 60:2253-2257, 2000 45. Koyanagi K, Kuo C, Nakagawa T, et al: Multimarker quantitative real-time PCR detection of circulating melanoma cells in peripheral blood: Relation to disease stage in melanoma patients. Clin Chem 51:981-988, 2005 46. Voit C, Kron M, Rademaker J, et al: Molecular staging in stage II and III melanoma patients and its effect on long-term survival. J Clin Oncol 23:1218-1227, 2005 47. Osella-Abate S, Savoia P, Quaglino P, et al: Tyrosinase expression in the peripheral blood of stage III melanoma patients is associated with a poor prognosis: A clinical follow-up study of 110 patients. Br J Cancer 89:1457-1462, 2003[CrossRef][Medline] 48. Szenajch J, Jasinski B, Synowiec A, et al: Prognostic value of multiple reverse transcription-PCR tyrosinase testing for circulating neoplastic cells in malignant melanoma. Clin Chem 49:1450-1457, 2003 49. Palmieri G, Ascierto PA, Cossu A, et al: Detection of occult melanoma cells in paraffin-embedded histologically negative sentinel lymph nodes using a reverse transcriptase polymerase chain reaction assay. J Clin Oncol 19:1437-1443, 2001 Submitted June 22, 2005; accepted March 16, 2006. Related Correspondence
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test for association, omitting unknown categories. P values < .05 were considered significant, but since many statistical tests were done for this article, P values between .01 and .05 should be considered marginally significant. All analyses were performed with GraphPad Prism Software (GraphPad Software Inc, San Diego, CA) and JMP software (SAS Institute Inc, Cary, NC). 
2 mm3 of each SLN was tested. The portion of the SLN analyzed by PCR simply may not have contained micrometastases, although this is nearly identical to the percentage of H and E/IHC- and PCR-positive patients (10.2%) who have developed recurrent disease. Any sampling method is just that—a sampling—and even with serial sectioning and IHC used in this study, only a small portion of each node was tested histologically or by RT-PCR. Whether sampling a larger portion of each node would have resulted in different results is uncertain, yet the fact that more than 40% of patients had RT-PCR evidence of melanoma cells in the SLN argues against the likelihood that a larger sample would have resulted in clinically applicable data. 
