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Journal of Clinical Oncology, Vol 22, No 19 (October 1), 2004: pp. 3989-3996 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.03.052 Serial Follow-Up and the Prognostic Significance of Reverse Transcriptase-Polymerase Chain Reaction—Staged Sentinel Lymph Nodes From Melanoma PatientsFrom the Departments of Surgery and Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY Address reprint requests to Udai S. Kammula, MD, Surgery Branch, National Cancer Institute, Building 10, Room 2B04, 10 Center Dr MSC 1502, Bethesda, MD 20892-1502; e-mail: udai_kammula{at}nih.gov
PURPOSE: Reverse transcriptase-polymerase chain reaction (RT-PCR) may provide an extremely sensitive method for detection of occult nodal disease. We evaluated the role of a single-marker RT-PCR assay for tyrosinase mRNA in the detection of melanoma sentinel lymph node (SLN) metastases and correlated the results with long-term clinical outcome. PATIENTS AND METHODS: One hundred twelve patients who underwent SLN biopsy for melanoma were prospectively analyzed. SLNs were bivalved, with half of each specimen evaluated by histologic methods and the other half evaluated by nested RT-PCR for tyrosinase. RESULTS: Fifteen patients (13%) had histologically positive SLNs, all of whom were also positive by RT-PCR (HISTO+/PCR+). Thirty-nine patients (35%) had SLNs that were negative by both histology and RT-PCR (HISTO–/PCR–). Fifty-eight patients (52%) were histologically negative but upstaged with a positive RT-PCR result (HISTO–/PCR+). Initially, at a median follow-up of 42 months, recurrence rates among the three cohorts were statistically different (HISTO+/PCR+, 53%; HISTO–/PCR+, 14%; and HISTO–/PCR–, 0%). However, at a longer median follow-up (67 months), recurrence rates for the HISTO–/PCR+ (24%) and HISTO–/PCR– (15%) groups were no longer statistically different (P = .25). The median time to relapse between the HISTO–/PCR+ and HISTO–/PCR– groups differed by 10 months (31 v 41 months, respectively). CONCLUSION: With extended follow-up of patients with histologically negative SLNs, detection of submicroscopic disease by tyrosinase RT-PCR does not define a subgroup that is at higher recurrence risk when compared with patients with RT-PCR–negative SLNs. Future studies evaluating molecular staging will require approximately 5 years of median follow-up to accurately define outcome for patients with occult melanoma metastases.
The presence of regional lymph node metastases is the most significant prognostic factor in patients with early-stage melanoma.1 Once nodal metastases are detected, the 5-year survival rate is reduced to less than 50%. The introduction of lymphatic mapping and sentinel lymph node (SLN) identification has proven to be an accurate technique to predict the pathologic status of regional lymph node basins.2 Furthermore, SLN histologic status has been shown to be the most significant predictor of survival in melanoma patients who present without clinical evidence of nodal metastasis.3 As a consequence, this minimally morbid and cost-effective technique has become the standard approach to stage patients with early melanoma and to define a cohort that may potentially benefit from early therapeutic intervention. The ideal methodology to evaluate SLN tissue, both to optimize sensitivity and specificity for the detection of occult metastases and to provide clinical relevance, is currently unclear. Conventional histologic examination with hematoxylin and eosin (HE) staining applied to bisected SLNs can be enhanced by using immunohistochemical (IHC) techniques and serial sectioning of the node if the initial evaluation fails to reveal evidence of metastatic disease. Enhanced histologic examination has been shown to improve the sensitivity for detecting micrometastatic deposits when compared with conventional histologic analysis.4-6 Nevertheless, histologic examination (HE and/or IHC) underestimates the number of melanoma patients who will develop recurrent disease. Recent SLN studies consistently demonstrate that 10% to 15% of patients with histologically negative SLNs will have clinical recurrences.7-10 Concerns regarding the sensitivity limitations of histologic analysis, as well as potential sampling error, have led to the application of molecular detection techniques in the analysis of SLNs. Reverse transcriptase-polymerase chain reaction (RT-PCR) can be used to identify mRNA transcripts of single or multiple target genes that are expressed by tumor cells. A common target has been the gene encoding tyrosinase, a key enzyme in the biosynthesis of melanin, which is found predominately in normal melanocytes and melanoma cells. RT-PCR amplification and recognition of such target genes can serve as a highly sensitive surrogate for the identification of melanoma tumor cells and, thus, enable greater detection of occult disease than conventional histologic techniques.11-15 In addition, a more comprehensive sample of the SLN can be efficiently processed when compared with the laborious step sectioning needed with enhanced histologic analysis. In prospective cohort studies, patients with histologically negative SLNs who have been upstaged by positive tyrosinase or multimarker RT-PCR assays have been reported to have a statistically worse outcome when compared with patients who have histologically negative SLNs with no detectable RT-PCR evidence of metastases.16-20 In fact, the remarkably low recurrence rate reported for patients with RT-PCR–negative SLNs (low false-negative rate) has prompted many to advocate that the strength of these RT-PCR assays may be their negative predictive value and the identification of a clinically significant low-risk prognostic cohort. Many of the conclusions from these RT-PCR studies, however, must be considered in the context of their limited duration of median follow-up (range, 19 to 42 months).16-20 This study was performed to evaluate the utility of a single-marker RT-PCR assay for tyrosinase mRNA in the detection of occult SLN metastases in the context of long-term follow-up. We correlated the results of this assay with clinical outcome from a single cohort of melanoma patients at an initial median follow-up of 42 months and, again, at 67 months. This sequential design attempts to validate previous studies that have had a median follow-up of 42 months or less and to examine the durability of these findings with longer follow-up. Differences in rates of recurrence, patterns of recurrence, and overall survival were serially examined. Such an analysis may help define the necessary duration of follow-up that is needed to interpret molecular staging studies of minimal volume disease (micrometastases).
Patients and Specimen Collection One hundred twelve patients with American Joint Committee on Cancer (fifth staging edition) stage I or II primary cutaneous melanoma and clinically negative lymph nodes were enrolled and evaluated between October 1996 and November 1999. This report represents the long-term follow-up of a cohort of patients that was originally presented in abstract form in 2001.21 Patients consented to enrollment in a prospective investigational protocol that was approved by the Institutional Review Board of the Memorial Sloan-Kettering Cancer Center. Patient information, including clinical characteristics, pathologic analysis, and follow-up, was prospectively entered into the melanoma database. All patients underwent preoperative lymphoscintigraphy with the intradermal injection of 99mTc-sulfur colloid (400 µCi) on the day of surgery and intraoperative intradermal injection of 1% isosulfan blue dye (Lymphazurin 1%; Hirsh Industries Inc, Richmond, VA). Then, all patients underwent wide local excision of their primary melanoma with selective lymphadenectomy performed using a standard clinical protocol at our institution.10 Promptly after excision, the SLNs were bivalved. One half of the specimen was snap frozen at –80°C and stored with a nonidentifying code for later tyrosinase RT-PCR analysis in a blind fashion. The other half of each SLN was formalin fixed and paraffin embedded for standard pathologic examination. At our institution, conventional histologic examination of SLNs included HE staining of the bisected SLNs. Beginning in late 1997, SLNs were routinely subjected to HE staining of serial sectioned specimens and IHC staining (S-100 and HMB-45) in the event that the initial HE staining of the specimen did not reveal evidence of metastatic disease. Patients found to have histologic evidence of SLN metastases underwent a complete lymph node dissection of that basin. Appropriate clinical conduct prevents retrospectively performing step sectioning and IHC on SLNs from patients who had originally been found to have negative SLNs with conventional histologic examination and who are still free of recurrence. No clinical decisions were based only on the results of the RT-PCR assays.
Nested RT-PCR for Tyrosinase RT-PCR for ß-actin was performed in all cases to confirm the integrity of the RNA extracted from SLNs. The assay used 400 to 800 ng of RNA from each sample in a single-step RT-PCR reaction using previously published primers and PCR conditions.25 The 154-bp PCR product generated from actin mRNA was detected by agarose gel electrophoresis.
Negative Controls
Follow-Up Evaluation
Statistical Analysis
Clinical and primary tumor characteristics for the 112 patients are listed in Table 1. The cohort had a median age of 52 years, with a slight male predominance. The majority of primary melanomas were located on an extremity, followed by the trunk. The median thickness of these tumors was 2.2 mm, with 84 (75%) being of intermediate depth (> 1 and  4 mm).
During this study period at our institution, histologic analysis of SLNs underwent a transition from conventional histologic examination with HE staining of the bisected SLN to the current method of HE staining of serial-sectioned specimens and IHC staining in the event that the initial HE staining of the specimen did not reveal evidence of metastatic disease. Conventional HE analysis was performed in 60 of the patients (54%), whereas step sectioning, HE, and IHC were performed in the remaining 52 patients (46%). Table 2 compares the primary tumor characteristics, pathologic results, and clinical outcome of these two groups. Both histologic groups had similar primary tumor characteristics, with a median thickness of 2.2 mm and a similar percentage of ulcerated primary tumors. The incidence of positive nodes detected by conventional HE analysis (15%) was not statistically different when compared with the current method of step sectioning and IHC (12%, P = .6); similarly, there was no difference in the associated rate of recurrence between the two groups (23% v 31%, respectively; P = .4) at an overall median follow-up of 67 months. As a combined histologic cohort of both methods, the overall incidence of positive nodes was 13% (15 of 112 patients), and the overall recurrence rate was 27% (30 of 112 patients) with a median follow-up of 67 months. RFS and DSS were significantly higher in patients with histologically negative SLNs than in patients with histologically positive SLNs, as shown in Figure 1.
Molecular marker analyses of SLN specimens were performed after all 112 patients had been confirmed to have at least one SLN with good-integrity RNA by RT-PCR amplification of actin mRNA. Tyrosinase mRNA was identified in SLNs from 73 patients (65%). Three groups of patients were defined by the histologic and molecular characteristics of their SLNs (Table 3). One group included 15 patients with histologically positive nodes, all of whom were also positive by RT-PCR analysis (HISTO+/PCR+ group). Another group included 39 patients (35%) who had SLNs that were negative by both histology and RT-PCR (HISTO–/PCR– group). The final group included 58 patients (52%) whose SLNs were histologically negative but were upstaged with a positive RT-PCR result (HISTO–/PCR+ group). Primary tumor median thickness for both the HISTO–/PCR– and HISTO–/PCR+ groups was 2.1 mm, whereas the HISTO+/PCR+ group had a median thickness of 2.4 mm. There was no significant difference in percentage of ulcerated primary tumors in these groups.
Recurrence and survival characteristics at the first follow-up period (median, 42 months) are listed in Table 4. There were eight recurrences (53%) in the HISTO+/PCR+ group. There were no recurrences in the HISTO–/PCR– group. Patients in the upstaged HISTO–/PCR+ group had eight recurrences (14%) and had a statistically worse RFS compared with the HISTO–/PCR– group (log-rank test, P = .02; Fig 2A). Analysis of the site of first recurrence in the HISTO–/PCR+ group showed that six of the eight patients had either nodal or systemic disease sites. There was no difference in DSS between the HISTO–/PCR– group and the HISTO–/PCR+ group (log-rank test, P = .16; Fig 2B).
Recurrence and survival characteristics at the subsequent follow-up period (median, 67 months) are listed in Table 5. There were two additional recurrences in the HISTO+/PCR+ group, resulting in 10 total recurrences (67%). The HISTO–/PCR+ group had six additional recurrences (24%), and the HISTO–/PCR– group had six new recurrences (15%). Ten of the 14 patients with later recurrences had primarily systemic sites of failure. At this 67-month follow-up, RFS for the HISTO–/PCR+ group was no longer statistically different when compared with the HISTO–/PCR– group (log-rank test, P = .25; Fig 2C); although, the median time to failure between the groups did differ by 10 months (31 v 41 months, respectively). There was no difference in DSS between the HISTO–/PCR– group and the HISTO–/PCR+ group (log-rank test, P = .35; Fig 2D) at the longer follow-up.
In recent years, it has become clear that SLN histologic status is the most powerful predictor of survival in patients with early-stage melanoma.3 Nevertheless, conventional (HE) and enhanced (IHC) histologic analysis underestimates the number of patients who present with recurrent disease.7-10 As a consequence, significant effort has focused on improving the sensitivity in detection of lymph node micrometastases by using molecular techniques. In 1991, Smith et al23 pioneered contemporary molecular staging of melanoma with the use of RT-PCR to detect tyrosinase mRNA in the peripheral blood of melanoma patients. Since then, the sensitivity of RT-PCR for tyrosinase transcripts has been estimated at a detection limit of one tumor cell in 106 to 107 background cells.12 It has been suggested that the application of RT-PCR to the examination of SLNs may reduce the inherent sampling error of routine histologic examination and may provide an extremely sensitive method of staging these lymph nodes. Although RT-PCR studies of SLNs support an improvement in sensitivity, it seems to come at the cost of increased false-positives and, thus, diminished specificity when compared with histologic analysis. False-positives in tyrosinase mRNA analysis may arise from assay contamination, illegitimate transcription, or legitimate sources such as tyrosinase-expressing Schwann cells and benign nevus cells. We have previously reported a false-positive rate of 11% using nested RT-PCR for tyrosinase in a group of 18 control lymph nodes from healthy donors.14 Despite these potential pitfalls, several investigators have reported significant prognostic value for RT-PCR in conjunction with histologic analysis of SLNs (Table 6). Using nested RT-PCR for tyrosinase mRNA, Shivers et al16 originally identified a cohort of patients who had histologically negative SLNs but who were positive for tyrosinase by RT-PCR. This upstaged group had an intermediate prognosis that was worse than the group that was negative by both histology and PCR. As shown in Table 6, other groups have confirmed this finding with demographically similar cohorts of patients.16-20 Furthermore, the HISTO–/PCR– group was found to have a significantly lower rate of recurrence and was considered a clinically privileged prognostic cohort. Although these studies have suggested that the presence of submicroscopic disease detected by RT-PCR techniques is clinically relevant, their conclusions have been based on limited duration of follow-up (range, 19 to 42 months). The current study was designed to examine the impact of longer duration of follow-up on the prognostic relevance of a molecular assay in the detection of occult SLN metastases in melanoma patients.
Similar to the other studies listed in Table 6, we evaluated the clinical outcome of three groups of patients defined by the histologic and molecular characteristics of their SLNs. At a median follow-up of 42 months, we, indeed, found that the upstaged HISTO–/PCR+ cohort had a worse RFS when compared with the HISTO–/PCR– group. In fact, the comparability of recurrence rates between the current series, calculated at the 42-month period, and those previously published with equal or less follow-up is remarkable. However, with an additional 2-year median observation period, our patients were found to have late recurrences that were predominately found in the HISTO–/PCR– group. At a median follow-up of 67 months, there was no longer a statistical difference in RFS among the histologically negative patients who were stratified by RT-PCR. Interestingly, although the probability of recurrence was not statistically different, the median time to recurrence between the HISTO–/PCR+ group and the HISTO–/PCR– group differed by 10 months (31 v 41 months, respectively). These findings are consistent with prior observations of time to initial recurrence in melanoma patients, in which the median duration to relapse was substantially longer in earlier-stage patients.25 However, our findings contradict other published reports that state that the main benefit of RT-PCR analysis is its negative predictive value and, thus, the ability to stratify patients with histologically negative SLNs into a distinct low-risk group based on a negative RT-PCR result. This study shows that the HISTO–/PCR– group has a defined long-term failure rate that is not different from the HISTO–/PCR+ group, with the exception of their time to recurrence. This finding became apparent only after the extended follow-up that was performed in this study. Although there is no definitive explanation for the observed late recurrences in the RT-PCR–negative group (ie, false-negatives), we present three possible hypotheses. The first theory suggests that tyrosinase RT-PCR is stratifying SLN micrometastases by burden of disease and not by the absolute presence or absence of occult nodal disease. Thus, low-volume micrometastases may exist in the HISTO–/PCR– group and would have a delayed presentation of clinically detectable recurrence when compared with higher volume micrometastases in the HISTO–/PCR+ group. This theory is compatible with clinical observations of macroscopic nodal metastases in which nodal tumor burden is associated with risk of recurrence.1 Furthermore, because no therapeutic intervention to the nodal basin was based on the RT-PCR findings in this study, both groups eventually should showed equal long-term RFS, which was indicated by the merger of their RFS curves on Kaplan-Meier analysis (Fig 2C). This theory, however, does not satisfyingly explain the observation that the majority of late relapses were systemic and not nodal. The second theory suggests that failures in the PCR-negative group could have resulted from inherent assay limitations. For example, if the nodal metastases lacked detectable tyrosinase expression, the assay would erroneously give a false-negative evaluation in the presence of tumor cells. Another technical limitation stems from potential sampling error, given that only half of the SLN was evaluated by the molecular assay. It is possible that micrometastases were in the portion of the node that was submitted for routine pathologic analysis and not detected by histologic methods. The third theory suggests unique biology and the possibility that these failures arose from direct hematogenous spread from the primary lesion, which bypassed the draining nodal station. This hypothesis may be supported by the observed pattern of first recurrence among the HISTO–/PCR– group, in which four of the six late recurrences were systemic. Future studies will need to delineate the role of these theories with respect to our observed findings. In the current study, our cohort could not be evaluated by a uniform histologic approach. The evolution of IHC and step sectioning as standard technique at our institution occurred in mid-1997. Given the prospective nature of our study and ongoing clinical follow-up of patients treated before this transition, we have observed patients who could not ethically be retrospectively evaluated by our current histologic approach. Analysis of the primary tumor characteristics, incidence of positive SLNs, and the associated recurrence rates between our conventional and current histologic approaches showed no differences (Table 2). Thus, we believe these groups can legitimately be combined for the purpose of this study. We do not attribute our new findings to this decision or possibly other methodologic differences between our study and the other published series. Substantiating this premise are the striking similarities between our results at the 42-month follow-up period and those of the other studies. The additional period of follow-up in this study seems to be the main variable in identifying the late recurrences. This study focused only on tyrosinase mRNA expression as a marker of metastases. Additional melanoma-related molecular markers include transcripts for GP100, MART-1, TRP-1, TRP-2, and members of the MAGE family. As single markers, these, in general, have proven less sensitive than tyrosinase because of highly variable or deficient expression in a significant percentage of metastatic lesions. Reports of multiple marker assays to improve both sensitivity and specificity have been published,26 and prospective evaluation is underway. To further improve the specificity of standard qualitative RT-PCR assays, real-time quantitative RT-PCR assays are being developed, which may allow for the establishment of a detection threshold above which sensitivity for clinically significant micrometastases is optimized and false-positives are minimized. Although these techniques may prove to be more accurate in the detection and characterization of occult metastases, we conclude from the results of this analysis that future studies evaluating molecular staging will necessitate suitable long-term median follow-up (approximately 5 years) to provide clinically relevant information to define outcome for patients with occult melanoma metastases.
The authors indicated no potential conflicts of interest.
Authors’ disclosures of potential conflicts of interest are found at the end of this article.
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3. Gershenwald JE, Thompson W, Mansfield PF, et al: Multi-institutional melanoma lymphatic mapping experience: The prognostic value of sentinel lymph node status in 612 stage I or II melanoma patients. J Clin Oncol 17:976-983, 1999 4. Cochran AJ, Wen DR, Herschman HR: Occult melanoma in lymph nodes detected by antiserum to S-100 protein. Int J Cancer 34:159-163, 1984[Medline] 5. Messina JL, Glass LF, Cruse CW, et al: Pathologic examination of the sentinel lymph node in malignant melanoma. Am J Surg Pathol 23:686-690, 1999[CrossRef][Medline] 6. Yu LL, Flotte TJ, Tanabe KK, et al: Detection of microscopic melanoma metastases in sentinel lymph nodes. Cancer 86:617-627, 1999[CrossRef][Medline] 7. Gershenwald JE, Colome MI, Lee JE, et al: Patterns of recurrence following a negative sentinel lymph node biopsy in 243 patients with stage I or II melanoma. J Clin Oncol 16:2253-2260, 1998[Abstract]
8. Gadd MA, Cosimi AB, Yu J, et al: Outcome of patients with melanoma and histologically negative sentinel lymph nodes. Arch Surg 134:381-387, 1999 9. Essner R, Conforti A, Kelley MC, et al: Efficacy of lymphatic mapping, sentinel lymphadenectomy, and selective complete lymph node dissection as a therapeutic procedure for early-stage melanoma. Ann Surg Oncol 6:442-449, 1999[Abstract] 10. Clary BM, Brady MS, Lewis JJ, et al: Sentinel lymph node biopsy in the management of patients with primary cutaneous melanoma: Review of a large single-institutional experience with an emphasis on recurrence. Ann Surg 233:250-258, 2001[CrossRef][Medline] 11. Goydos JS, Ravikumar TS, Germino FJ, et al: Minimally invasive staging of patients with melanoma: Sentinel lymphadenectomy and detection of the melanoma-specific proteins MART-1 and tyrosinase by reverse transcriptase polymerase chain reaction. J Am Coll Surg 187:182-188, 1998[CrossRef][Medline] 12. Wang X, Heller R, VanVoorhis N, et al: Detection of submicroscopic lymph node metastases with polymerase chain reaction in patients with malignant melanoma. Ann Surg 220:768-774, 1994[Medline]
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16. Shivers SC, Wang X, Li W, et al: Molecular staging of malignant melanoma: Correlation with clinical outcome. JAMA 280:1410-1415, 1998
17. Bostick PJ, Morton DL, Turner RR, et al: Prognostic significance of occult metastases detected by sentinel lymphadenectomy and reverse transcriptase-polymerase chain reaction in early-stage melanoma patients. J Clin Oncol 17:3238-3244, 1999 18. Blaheta HJ, Ellwanger U, Schittek B, et al: Examination of regional lymph nodes by sentinel node biopsy and molecular analysis provides new staging facilities in primary cutaneous melanoma. J Invest Dermatol 114:637-642, 2000[CrossRef][Medline] 18. Li W, Stall A, Shivers SC, et al: Clinical relevance of molecular staging for melanoma: Comparison of RT-PCR and immunohistochemistry staining in sentimel lymph nodes of patients with melanoma. Ann Surg 231:795-803, 2000[CrossRef][Medline] 19. Goydos JS, Patel KN, Shih WJ, et al: Patterns of recurrence in patients with melanoma and histologically negative but RT-PCR-positive sentinel lymph nodes. J Am Coll Surg 196:196-204, 2003[CrossRef][Medline]
20. 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 21. Ghossein RA, Nissan A, Stojadinovic A, et al: Prognostic value of tyrosinase mRNA reverse transcription-polymerase chain reaction in the evaluation of melanoma sentinel lymph nodes. Presented at 37th Annual Meeting of Am Soc Clin Oncol, San Francisco, CA, May 12-15, 2001
22. Ghossein RA, Coit D, Brennan M, et al: Prognostic significance of peripheral blood and bone marrow tyrosinase messenger RNA in malignant melanoma. Clin Cancer Res 4:419-428, 1998 23. Smith B, Selby P, Southgate J, et al: Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 338:1227-1229, 1991[CrossRef][Medline] 24. Ben-Ezra J, Johnson DA, Rossi J, et al: Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction. J Histochem Cytochem 39:351-354, 1991[Abstract] 25. McCarthy WH, Shaw HM, Thompson JF, et al: Time and frequency of recurrence of cutaneous stage I malignant melanoma with guidelines for follow-up study. Surg Gynecol Obstet 166:497-502, 1988[Medline] 26. Taback B, Morton DL, O’Day SJ, et al: The clinical utility of multimarker RT-PCR in the detection of occult metastasis in patients with melanoma. Recent Results Cancer Res 158:78-92, 2001[Medline] Submitted March 5, 2004; accepted July 26, 2004.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test for categoric outcome was used to compare the difference between groups. Relapse-free survival (RFS) was calculated from the date of SLN biopsy to the date of last follow-up evaluation or to the date of first recurrence. Disease-specific survival (DSS) was calculated from the date of SLN biopsy to the date of melanoma-related death. RFS and DSS curves were constructed using the Kaplan-Meier product-limit method, and differences between cohorts were tested with log-rank statistics. In all statistical analyses, P < .05 was considered significant. 
