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Originally published as JCO Early Release 10.1200/JCO.2003.01.063 on August 11 2003 © 2003 American Society for Clinical Oncology Prediction of Disease Outcome in Melanoma Patients by Molecular Analysis of Paraffin-Embedded Sentinel Lymph Nodes
From the Department of Molecular Oncology, Department of Pathology, Division of Biostatistics, and Department of Surgical Oncology, John Wayne Cancer Institute, St John’s Health Center, Santa Monica, CA. Address reprint requests to Bret Taback, MD, Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404; e-mail: tabackb{at}jwci.org.
Purpose: A significant number of patients who develop recurrence after a histopathologically negative sentinel lymph node (SLN) biopsy will demonstrate occult metastases on re-evaluation of the SLNs with serial sectioning and immunohistochemistry. Reverse transcriptase polymerase chain reaction (RT-PCR) has been evaluated to improve disease staging and avoid false-negative findings in fresh or frozen-section SLNs. The purpose of this study was to develop a multimarker RT-PCR assay for assessing melanoma patients’ archived paraffin-embedded (PE) SLNs. Patients and Methods: Archived PE histopathologically positive (n = 37) and negative (n = 40) SLNs from patients with primary melanoma were analyzed using a semiquantitative multimarker RT-PCR assay. Results: Marker expression in histopathologically positive and negative SLNs were as follows: 89%, 92%, 35%, and 43% (positive) and 40%, 33%, 5%, and 13% (negative) for tyrosinase, melanoma antigen recognized by T cells-1, tyrosinase-related protein-1, and tyrosinase-related protein-2, respectively. Twenty-five percent of histopathologically negative SLN patients were upstaged using at least two markers. Of these, 80% developed a recurrence. Furthermore, at a median follow-up of 55 months, patients with histopathologically negative SLNs who expressed zero or one marker had a significantly improved disease-free (P < .002) and overall (P < .03) survival versus those expressing two or more markers. Conclusion: These findings demonstrate the feasibility of a multimarker RT-PCR assay for evaluating archived PE SLNs. More significantly, identification of molecular risk factors can be detected in histopathologically negative SLNs for distinguishing early-stage melanoma patients with a worse prognosis.
THE MOST frequent site of early melanoma metastases is the regional tumor-draining lymph node basin. The technique of lymphatic mapping and sentinel lymph node (SLN) identification has proven highly accurate for predicting the pathologic status of the regional lymph node basin without the added expense and morbidity associated with a conventional complete lymphadenectomy.1,2 This approach also allows for a more focused, efficient, and comprehensive search for occult metastases that may be prognostically significant.3,4 Immunohistochemical (IHC) techniques using anti–S-100 and anti–HMB-45 antibodies have demonstrated a 10% to 30% improved sensitivity for identifying micrometastases compared with conventional hematoxylin and eosin (HE) staining.5–8 IHC techniques permit facile pathologic review of lymph node slides and rapid identification of occult tumor cells against a background of lymphocytes. These findings are important because the most significant prognostic factor in patients with early-stage melanoma, American Joint Committee on Cancer stage I and II, is the presence of lymph node metastases.9 In addition, the most common site of first recurrence in patients with negative SLNs is the regional nodal basin, and studies re-evaluating these SLNs with serial sectioning and/or IHC have demonstrated occult metastases in a significant number of these patients.10,11 More recently, reverse transcriptase polymerase chain reaction (RT-PCR) methods have been used to detect subclinical melanoma metastases.12–15 This technique has been shown to upstage an additional 8% to 52% of patients with histopathologically negative SLNs when compared with HE and IHC staining.16–20 Moreover, clinical studies have demonstrated a significantly decreased disease-free survival (DFS) among patients whose SLN was RT-PCR–positive for melanoma marker expression when compared with patients with SLNs that were RT-PCR–negative.17,18 These findings demonstrate the enhanced sensitivity of RT-PCR for detecting clinically significant submicroscopic SLN metastases compared with histopathologic staining with HE and IHC. An additional advantage with RT-PCR is that it can be rapidly and reliably performed. RT-PCR also circumvents the potential for erroneous results associated with processing artifacts caused by the haste for an expeditious diagnosis during intraoperative frozen section analysis with immediate IHC for detecting micrometastases.8 However, these studies required sacrificing a portion of the SLN for RT-PCR and thus diverting valuable tissue from pathologic inspection. A more desirable approach is to evaluate paraffin-embedded (PE) tissues after pathologic review is performed. The use of frozen or fresh tissues for RT-PCR requires time-consuming processing regimens, including extensive efforts in storing frozen tissue, and limits investigations to prospective studies. RT-PCR using archived PE tissues would allow assessment of multiple tissue samples from patients, with well-defined end points for clinical correlation and a more rapid evaluation of a marker’s utility. In this study, we investigated the feasibility of a multimarker (MM) RT-PCR assay to detect melanoma marker mRNA in PE melanoma metastases. Moreover, to evaluate the clinicopathologic utility of this assay, we assessed archived histopathologically negative and positive SLNs from patients with clinical early-stage melanoma to determine the ability of the assay to upstage patients. RT-PCR results for the detection of melanoma metastases were compared to the HE and/or IHC findings in the SLNs and correlated with patient clinical, pathologic, and disease outcome data.
Specimen Collection RNA isolated from seven established melanoma cell lines (MA, MB, MC, MD, ME, MF, and MG) from the John Wayne Cancer Institute (Santa Monica, CA) was assessed for marker expression by RT-PCR and optimization of the electrochemiluminescence (ECL) detection assay. PE normal (negative for cancer) lymph nodes (n = 10) and normal tonsil tissues (n = 15) served as negative controls for all RT-PCR reactions. Forty-five PE tissues of melanoma metastases were evaluated for mRNA presence using a panel of four melanoma-associated antigen markers, tyrosinase (Tyr), melanoma antigen recognized by T cells-1 (MART-1), tyrosinase-related protein-1 (TRP-1), and TRP-2, using an RT-PCR semiquantitative ECL detection system (referred to as the MM RT-PCR assay).21–23 After successful demonstration of the assay in archived metastatic tumor tissue, PE blocks containing SLNs from 77 patients were obtained from the Department of Pathology at Saint John’s Medical Center (Santa Monica, CA). All studies were approved by the Saint John’s Medical Center Institutional Review Board Committee. A pathologic diagnosis of primary cutaneous melanoma was made for all patients, and no clinical evidence of lymph node metastases was present at the time of SLN dissection. Thirty-seven patients had a histologic diagnosis of SLN metastases, whereas 40 patients had no evidence of tumor cells in their SLN by HE and/or IHC. Patient demographics are listed in Table 1  . Clinical history on all patients was available from the melanoma computer database at the John Wayne Cancer Institute.
Histopathologic evaluation of formalin-fixed PE SLNs was performed such that several 4-µm-thick sections were cut at two levels separated by a 200-µm interval and stained with conventional HE. Immunostaining was performed on the adjacent cut sections with antibodies to S-100 and HMB-45 proteins. For MM RT-PCR analysis, 10 sections of 10-µm-thick PE metastatic melanomas, SLNs, normal lymph nodes, and tonsil tissues were deparaffinized with xylene. A modification of the Paraffin Block RNA Isolation Kit procedure (Ambion, Austin, TX) was used. After proteinase K digestion, RNA was solubilized in guanidinium-based buffer and separated with phenol-chloroform. For improved precipitation of RNA in isopropanol, Pellet Paint (Novagen, Madison, WI) was added. The total RNA isolated was quantified by ultraviolet spectrophotometry. The integrity of all RNA samples was verified by performing an RT-PCR assay for the housekeeping gene, beta2-microglobulin.
MM RT-PCR Assay In the ECL assays, the sense primer was biotinylated (Sigma Genosys, Woodland, TX) for bead capture and the antisense primer was labeled with tris(2,2-bipyridine) ruthenium(II) (TBR; Midland Certified Reagent Co, Midland, TX) for detection. Specific primers were designed for the amplification of cDNA with primer sequences spanning at least one exon-exon region. Primer sets for tumor markers were designed for optimal activity using the ECL system. The ECL assay requires that the PCR products contain both the biotin and TBR for detection. Primer sequences consisted of biotin-5'-AGCTATCTACAAGATTCAGACC-3' and TBR-5'-AAGGAGCCATGACCAGATCCG-3' for Tyr, biotin-5'-GATCATCGGGACAG CAAAGTG-3' and TBR-5'-GTGGAGCATTGGGAACCAC AG-3' for MART-1, biotin-5'-ACTTTGTAACAGCACCGAGGA-3' and TBR-5'-AGCGACATCCTGTGGTTCA-3' for TRP-1, and biotin-5'-GATACATTATTAGGACCAGGA-3' and TBR-5'-AGATCTCTTTCCAGACACAAC-3'for TRP-2. The post-PCR double-stranded transcripts of specific genes were captured by M-280 streptavidin-coated Dynabeads (Dynal Biotech, Lake Success, NY), and the Origen Analyzer (IGEN, Bethesda, MD) was used to measure the ECL activity as previously described.24 Results were expressed as ECL units (ECL U), and positive samples were determined when the sample expressed a level of ECL U greater than the cutoff point. The cutoff point for determining sample positivity was three standard deviations above the mean ECL U of the PE normal lymph nodes and tonsil tissues assessed in each assay. For each assay, at least three positive controls (cell lines that tested positive for the respective markers), at least four PE normal lymph nodes or tonsil samples, and reagent negative controls (reagent alone without RNA or cDNA) were included. Each run contained its own set of controls to establish background levels of ECL U for the RT-PCR assay system, and all assays were repeated twice to verify results. To prevent contamination, tissue processing, RNA isolation, RT-PCR setup, and analysis of post–RT-PCR products were performed in separate rooms under strict standard operating procedures.23 The sensitivity limit was determined in a dilution study in which RNA isolated from PE melanoma tumor tissue was serially diluted 10-fold from 1 µg for RT-PCR assessment. Tyr and MART-1 can be consistently detected using 0.1 ng of RNA. TRP-1 and TRP-2 can be consistently detected using 1 ng of RNA.
Statistical Analysis
All four melanoma-associated antigen mRNA transcripts were expressed by the seven melanoma cell lines using ECL detection. Tyr, MART-1, TRP-1, and TRP-2 expression was detected in 56% to 89% of archived PE metastatic tumors (n = 45; Table 2 ). At least one melanoma marker could be detected in all metastatic tumor specimens, and 56% had three markers identified successfully. None of the cancer-negative PE lymph nodes (n = 10) and tonsil tissues (n = 15) expressed any of the melanoma markers. These results provided the basis for an optimal marker combination with adequate sensitivity and specificity to be used for assessing PE SLNs.
After successful demonstration of marker expression in metastatic tumors, archived PE SLNs from 77 patients with clinical early-stage cutaneous melanoma were assessed for the expression of the four melanoma markers. All patients underwent SLN dissection at the time of their primary diagnosis, and none had clinical evidence of regional lymph node metastases. Thirty-seven patients had histologic evidence of SLN metastases by conventional HE staining and/or IHC, and 40 patients’ SLNs were histopathologically negative (Table 3 ). Among the patients with histopathologically positive SLNs, 35 (95%) of 37 demonstrated at least one detectable marker, with MART-1 occurring most frequently in 34 (92%) of 37 patients’ SLN, followed by Tyr (89%), TRP-2 (43%), and TRP-1 (35%). At least two melanoma markers were detected in 86% of patients and almost one half of the patients expressed three or more markers. In two instances no melanoma markers were detected, resulting in a false-negative rate of 5%.
Among 40 patients with histopathologically negative SLN, MM RT-PCR detected at least one melanoma marker in 22 (55%) patients and two or more markers in 10 patients (25%; Table 3  ). Only four patients (10%) with histopathologically disease-free SLN had three or more markers. The most frequent mRNA marker identified was Tyr, which occurred in 16 (40%) of 40 patients without histopathologic evidence of SLN metastases; this was followed by MART-1 (33%), TRP-2 (13%), and TRP-1 (5%).
Statistical analysis was performed to determine if the number or type of melanoma markers expressed correlated with any known patient clinical and pathologic prognostic parameters: sex, age, primary anatomic site, Clark level, Breslow thickness, and histopathologic SLN status. There was a significant association between increasing number of markers expressed and Breslow thickness (Spearman correlation, P < .01). Specifically, mean tumor Breslow thickness was significantly correlated with either Tyr or MART-1 expression (t test, P = .02 and P < .001, respectively). When thickness was categorized according to American Joint Committee on Cancer tumor (T classification) size, a significant correlation existed between TRP-2 or MART-1 detection and increasing tumor size (
Furthermore, when dichotomized by the number of positive markers (Table 1
Patients were observed clinically for a median of 55 months. Forty-four (57%) of 77 patients developed a melanoma recurrence. Univariate analysis revealed a significant correlation between the expression of single markers TRP-1 (P < .03), MART-1 (P < .002), and Tyr (P < .001) or any combination of two or more markers (P < .001) and DFS. When stratified by histopathologic SLN status, 23 (62%) of 37 patients with positive SLNs developed a melanoma recurrence, whereas 21 (53%) of 40 patients without SLN metastases developed a melanoma recurrence (Table 3
A Cox proportional hazards model was developed to correlate melanoma marker expression in the SLN with DFS while controlling for the following clinicopathologic prognostic parameters: sex, age, primary tumor site, Breslow thickness, and the histologic presence of SLN metastases. Multivariate analysis demonstrated Tyr (P = .029) or MART-1 (P = .047) marker expression was significantly associated with a decreased DFS, with risk ratios of 2.64 (95% CI, 1.10 to 6.32) and 2.43 (95% CI, 1.01 to 5.82), respectively. The risk of developing a recurrence in patients with SLNs expressing more than two markers increased by a factor of 2.51 (95% CI, 1.00 to 6.30; P < .05) over those patients with zero to one markers detected. In patients with SLNs deemed free of tumor cells by histopathology, a significantly greater number of patients who experienced disease recurrence demonstrated Tyr in their SLNs, with a risk ratio of 3.19 (95% CI, 1.23 to 8.27; P = .017; Fig 1
Twenty-three (29.9%) of 77 patients died as a result of metastatic melanoma. Overall survival was correlated with marker expression, and only Tyr was found to be significant in both univariate (P = .046) and multivariate analyses (P = .015). The combination of more than two markers expressed was associated with a significant decrease in overall survival for all patients in this study in univariate analysis (P = .045), but did not reach statistical significance in multivariate analysis. Patients with histopathologically negative SLNs who expressed Tyr had a higher risk of death as a result of recurrent melanoma than histopathologically negative SLN patients who did not express Tyr, with a relative risk of 6.14 (95% CI, 1.66 to 22.69; P < .007; Fig 3 ). Furthermore, among patients with negative SLNs by histopathology, those with more than two markers detected had a relative risk of death of 4.73 compared with patients with zero to one marker expressed (95% CI, 1.17 to 19.20; P < .03; Fig 4).
Because the presence of lymph node metastases remains the single most important prognostic factor for patients diagnosed with melanoma, precise staging of the tumor-draining lymph node basin is critical patient care information. Furthermore, these results may help stratify patients at increased risk for relapse who may then be eligible for adjuvant therapy trials. The SLN concept is based on the principle that lymphatic spread of solid tumors occurs in an organized manner through tumor-draining lymph nodes such that the most likely site of early metastases occurs in the first or sentinel of this lymph node hierarchy.1 Given this information, identification of the SLN should accurately predict the pathologic status of a tumor’s draining lymph node basin. This concept has been proven for a variety of solid neoplasms across many multi-institutional studies.25–27 This technique provides the pathologist with a limited number of lymph nodes with which to perform a more aggressive search for micrometastases. Thus, diagnosis with SLN identification is both more accurate and less costly because of the reduced time and materials associated with investigating all lymph nodes obtained from a conventional lymphadenectomy specimen. The presence of lymph node metastases is associated with a dramatic reduction in overall survival, up to 40% to 50% in most series. A variety of pathologic methods have been recommended to enhance detection of melanoma metastases. Serial sectioning and IHC have been shown to improve the identification rate of melanoma tumor cells in lymph nodes compared with routine HE staining.3,5 More recently, RT-PCR has shown a greater sensitivity for detecting submicroscopic metastases by amplifying gene transcripts associated with occult tumor cells from organ-specific malignancies.17,28–31 For melanoma, these studies have demonstrated a 10% to 44% upstaging by RT-PCR of SLNs reported negative by histopathologic examination.16–20 These findings are striking because outcome studies have shown a 13% to 25% recurrence rate in histopathologically negative, Tyr RT-PCR–positive SLN patients.17,18,20 Previously, RT-PCR studies involved the evaluation of frozen or fresh tissue for analysis, which may not always be available and requires added time, expense, and efforts for RNA processing and preserving. More importantly, this diverts the limited precious specimen from histopathologic processing and review, which currently remains the gold standard for patient diagnosis. MM RT-PCR assessment of PE SLNs circumvents these problems and provides a more practical and logistically convenient approach. This method permits assessment of multiple specimens at once and can be used for performing retrospective studies with well-defined patient populations and clinical outcomes for a better, more expeditious assessment of marker utility. As a result, this approach advances the integration of additional new markers when they are identified as being significant. Recently, the potential utility of molecular analysis for assessing archived PE specimens has been demonstrated in various cancers.32,33 Although SLN metastasis is associated with a poorer prognosis, most patients will have a negative SLN by histopathologic review. However, 10% to 15% will subsequently develop a recurrence. This prevalence will increase as more early-stage melanomas are diagnosed. Additional factors are needed to identify patients at risk for relapse. Gadd et al10 and Gershenwald et al11 re-evaluated histopathologically negative SLN from patients after melanoma recurrence using IHC and serial sectioning, and they were able to identify occult tumor cells in 33% and 43% of these patients, respectively. The purpose of this study was to determine whether a retrospective analysis of archived PE SLNs considered negative for tumor cells by HE and IHC from patients who experienced relapse within 5 years could identify occult metastases in comparison with those patients who did not experience recurrence. With the MM RT-PCR approach, at least one marker was identified from 35 (95%) of 37 histologically confirmed SLNs, demonstrating the accuracy of this assay. Only two false-negative results occurred, which demonstrates the feasibility of MM RT-PCR for assessing archived PE tissues. The mean age of the paraffin blocks assessed in this study was 5 years, and the oldest block that was positive for mRNA expression was in archive for 13 years. Of the patients with histopathologically negative SLN, 55% expressed one or more marker and 25% expressed two or more markers. Interestingly, we found a statistically significant increased expression of these markers from histopathologically negative SLN patients who developed a recurrence compared with those SLN-negative patients who did not develop a recurrence. In addition, patients with histopathologically negative SLN who expressed two or more markers had a DFS equivalent to those patients with positive SLN by histopathology. Similarly, overall survival was significantly reduced for patients with histopathologically negative SLN who expressed two or more melanoma markers. These findings support the utility of molecular techniques to identify submicroscopic disease, which we have shown to be of clinical significance. Important patient care decisions may be affected by the factor that patients with negative SLN by both histopathology and MM RT-PCR have a significantly more favorable prognosis. In summary, MM RT-PCR can be performed successfully to detect melanoma metastases in archived PE SLNs with excellent correlation to histopathologic tumor presence. More importantly, this study demonstrates the identification of molecular risk factors that can be detected in histopathologically negative SLNs to distinguish early-stage melanoma patients with a poorer prognosis.
The authors indicated no potential conflicts of interest.
Supported in part by National Institutes of Health grants NCI P01 CA29605 and P01 CA12582, and the Roy E. Coats Research Laboratories of the John Wayne Cancer Institute.
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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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ABSTRACT
INTRODUCTION
2 test (for categorical variables) were performed to compare the differences between the marker-positive and -negative groups. The correlation of marker expressions with DFS and overall survival was examined using the log-rank test. The Cox proportional hazards regression model also was developed to correlate survival with marker expression while the known prognostic factors, such as age, sex, primary site, Breslow thickness, and nodal status were adjusted. Kaplan-Meier survival curves also were plotted. Statistical analysis was performed using SAS (Version 8.2; SAS Institute, Cary, NC). 
