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© 2001 American Society for Clinical Oncology Detection of Occult Melanoma Cells in Paraffin-Embedded Histologically Negative Sentinel Lymph Nodes Using a Reverse Transcriptase Polymerase Chain Reaction AssayFrom the Institute of Molecular Genetics, National Research Council of Italy, Alghero (SS); the Institute of Pathology, University of Sassari, Sassari; the National Tumor Institute "Fondazione G. Pascale"; and the Department of Dermatology, Second University of Naples, Naples, Italy Address reprint requests to Giuseppe Palmieri, MD, Institute of Molecular Genetics, National Research Council of Italy, Alghero (SS), Casella Postale, 07040 Santa Maria La Palma (Sassari), Italy; email: gpalmieri{at}yahoo.com
PURPOSE: Detection of occult metastasis before the development of clinical disease could allow more accurate staging, appropriate follow-up procedures, and adjuvant therapies in patients with malignant melanoma (MM). The sentinel lymph node (SLN) has been proposed as a reliable predictor of metastatic disease in the lymphatic basin draining the primary melanoma. In this study, we screened both paraffin-embedded SLNs and peripheral-blood (PB) samples from MM patients at various stage of disease using a multimarker reverse transcriptase polymerase chain reaction (RT-PCR) assay. The prognostic significance of the presence of PCR-positive markers was also evaluated. PATIENTS AND METHODS: Total RNA was obtained from paraffin-embedded SLN sections and PB samples of 75 MM patients. RT-PCR was performed using tyrosinase and MelanA/MART1 as melanoma-associated markers. Radiolabeled PCR products were analyzed on denaturing polyacrylamide gels. RESULTS: Good sensitivity of the RT-PCR assay on archival tissues was demonstrated after comparison of RT-PCR results on frozen and paraffin-embedded SLNs from 16 MM patients. Significant correlation between the disease stage and marker expression in both PB and SLN samples was observed; the highest value was for patients who were positive for both markers in SLN (P = .006). Progression of disease was significantly associated with the total number of PCR-positive markers in both PB (P = .034) and SLN (P = .001) samples. CONCLUSION: Although sensitivity is lowered by the use of paraffin-embedded specimens, our data indicate that RT-PCR analysis of serial sections from archival SLNs may be helpful in improving detection of occult micrometastases, thus improving staging of patients with melanoma.
PROGNOSTIC CLASSIFICATION in malignant melanoma (MM) is currently based on histopathologic parameters of the primary tumor, such as Breslow’s tumor thickness and Clark’s level of invasion.1,2 However, development of regional node metastasis is able not only to change tumor staging but also to significantly affect patient survival, becoming the main prognostic factor in MM.3 Identification and subsequent surgical resection of occult metastasis before the development of clinical disease may improve survival in melanoma patients. In past years, selective lymphadenectomy has been introduced in the treatment of MM by using lymphatic mapping techniques (initially based on intradermic injection of vital blue dye, and then improved by radioguided methodology).4-7 The sentinel lymph node (SLN) dissection, which removes the first node(s) receiving melanoma cells from the primary tumor, has been recently proposed as an effective tool for the detection of occult MM metastasis.8 As reported by Morton et al,4 absence of melanoma cells in SLNs is a strong predictor that no other (nonsentinel) node in the draining lymphatic basin contains MM metastatic cells. Therefore, detection of melanoma cells in SLNs could significantly contribute to a more accurate survival prediction for MM patients, with the assessment of a more accurate staging, and to more appropriate follow-up procedures and/or more effective adjuvant therapies.9,10 Immunohistochemical (IHC) analysis of serial sections, using HMB-45 and S-100 antibodies, improves the detection of occult melanoma cells in surgically resected SLNs, compared with conventional hematoxylin and eosin staining alone.4,11 However, submicroscopic MM cells could be identified exclusively by application of highly sensitive molecular biology approaches.12,13 In particular, amplification of tissue-specific mRNA present in the tumor cells by reverse transcriptase polymerase chain reaction (RT-PCR) may be the main strategy for the detection of micrometastases in patients with solid tumors.14 RT-PCR assays using multiple mRNA markers are more inclusive than assays using single mRNA markers for detecting heterogeneous populations of occult metastatic melanoma cells in both peripheral blood (PB)15-17 and lymph nodes of MM patients.18 Previously, we performed RT-PCR analysis of PB samples from a large collection of patients with MM using tyrosinase, p97, MUC18, and MelanA/MART1 as markers.17 This assay was highly sensitive in detecting circulating MM cells, the presence of which was significantly correlated with the disease stage.17 To further evaluate the prognostic clinical utility of the multimarker RT-PCR assay in melanoma and to use our large collection of patient samples, we developed a protocol for amplification of tyrosinase and MelanA/MART1 mRNAs in paraffin-embedded samples of SLNs from MM patients. Taking into account published data,15-22 we selected these two markers based on their higher specificity for expression in cells of the melanocytic lineage or, mostly, in primary and metastatic melanomas. The aim of this study was to assess the sensitivity of a multimarker RT-PCR assay in detecting MM micrometastasis in both histologically proven tumor-negative SLNs and PB samples from a subset of patients with localized disease. This study also assessed sensitivity by comparison with the group of patients presenting IHC-positive lymph nodes, classified as American Joint Committee on Cancer (AJCC) stage III, and evaluated the prognostic significance of the presence of PCR-positive markers.
Sample Collection Patients with a histologically documented diagnosis of MM and no clinical evidence of regional or metastatic disease underwent the SLN dissection, as described by Morton et al.4 After giving informed consent, melanoma patients with disease stage recorded as IA, IB, IIA, or IIB (according to AJCC guidelines23) were enrolled onto the study. For histopathologic examination, hematoxylin and eosin staining and IHC were performed on adjacent sections of each formalin-fixed paraffin-embedded SLN. For IHC, 4-µm–thick sections were evaluated with antibodies to the HMB-45 and S-100 proteins. The SLN was considered negative if no melanoma cells were identified using both methodologies, and the result was confirmed by a similar evaluation of at least two additional paraffin-embedded sections (from a distant level, separated by approximately 50 µm). For RT-PCR analysis of paraffin-embedded SLNs, three to four immediately adjacent 8-µm–thick sections, and two to three 8-µm–thick nonadjacent sections (from a distant level, separated by approximately 50 µm) were processed to isolate total RNA. For 16 MM patients, frozen portions of SLNs were available and total RNA was obtained from five to six immediately adjacent 10-µm–thick sections cut on the cryostat. Blood samples were taken within 1 month after surgical treatment of both primary tumor and SLN for nonmetastatic melanoma patients and within 3 to 4 weeks after lymph node dissection for AJCC stage III patients (used as positive controls in this study). Clinical staging (medical history, physical examination, blood cell count, and blood biochemistry) and eventual current or past therapies were documented. Disease status was defined depending on the absence or presence of clinical melanoma at the moment of blood extraction, and disease progression was determined by a worsening in disease status (in terms of appearance of any recurrence). No clinical decisions were made based on the results of the RT-PCR assays. Patients had follow-up visits every 4 months after the diagnosis consisting of clinical history, physical examination, blood cell count, and blood biochemistry. Paraffin sections of 18 tumor-negative lymph nodes from patients with nonmelanoma malignancies (four colorectal carcinomas and 14 breast cancers) were used as negative controls for setting conditions for the RT-PCR assay.
Sample Preparation The protocol used for RNA extraction from paraffin-embedded tissues was a modification from Jackson et al.24 Briefly, paraffin sections were treated with 1 mL of xylene in polypropylene, stirred for 30 minutes at room temperature (RT), and centrifuged at 12,000 rpm for 20 minutes at RT. After washes with absolute and 70% ethanol, the pellet was air dried and resuspended in PK-GT lysis solution (0.5 mg/mL of proteinase K in GT [4 mol/L guanidine thiocyanate; 25 mmol/L of sodium citrate, pH 7.0; 0.5% sarkosyl; 0.1 mol/L 2-mercaptoethanol]). Samples were incubated at 45°C for 2 hours before adding 0.1 volume of 2 mol/L sodium acetate, pH 4.0; 0.5 volume of acid phenol (equilibrated, saturated with water); and 0.2 volume of chloroform/isoamyl alcohol (49/1). Samples were mixed well by vortexing, incubated on ice for 20 minutes, and centrifuged at 12,000 rpm for 20 minutes at 4°C. After precipitation of the supernatant with one volume of isopropanol for 2 hours at -20°C and centrifugation as above, the pellet was washed with absolute and 70% ethanol, resuspended in RNase-free distilled water, and stored at -80°C.
RT-PCR Assay Final products were diluted 1/1 in denaturing load buffer (95% formamide, 10 mmol/L of NaOH, 0.05% xylene cyanol FF, and 0.05% bromphenol blue) and denatured at 94°C for 5 minutes; 4 µL of this solution was loaded for electrophoresis onto a 6% polyacrylamide gel containing 7 mol/L of urea at 80W. The gel was vacuum-dried and exposed to Hyperfilm MP autoradiography film (Amersham, Buckinghamshire, United Kingdom) for 16 hours at RT. Each step of the RT-PCR procedure (RNA extraction, RT-PCR assay set-up, and PCR product analysis) was performed in a separate designated room to prevent cross-contamination because of the high sensitivity of the assay. In each RT-PCR assay, controls included cDNA from the human melanoma-derived cell line SK-MEL-28 (American Type Culture Collection) as an amplification positive control, PCR reagents and primers without template as a reaction negative control (to reveal abnormal PCR mixture contamination), and an amplification control for the housekeeping gene GAPDH (to facilitate quantitative and qualitative assessment of both RNA extraction and cDNA synthesis), as previously described.17 Nine samples that were negative for amplification of GAPDH mRNA were excluded from the study.
Statistical Analysis
In a previous report, we tested the sensitivity and specificity of a multiple-marker RT-PCR assay for detecting circulating melanoma-associated mRNA markers.17 Analysis of total cellular RNA obtained from PB of 235 patients with either localized or metastatic MM and from 41 negative controls (20 healthy subjects and 21 patients with different types of cancer) demonstrated that maximal values of sensitivity and specificity were reached by tyrosinase and MelanA/MART1.17 Therefore, we decided to evaluate the sensitivity and specificity of these two RT-PCR markers in detecting occult tumor cells in archival SLNs removed from melanoma patients. Paraffin sections from tumor-free lymph nodes obtained from 18 nonmelanoma cancer patients (four colorectal carcinomas and 14 breast cancers) were screened with tyrosinase and MelanA/MART1 markers to evaluate the specificity of the RT-PCR assay. None of the 18 control lymph nodes was positive for tyrosinase, whereas a faint PCR-positivity for MelanA/MART1 was detected in one (6%) specimen (data not shown). To assess the sensitivity of the RT-PCR assay on archival tissues, comparative amplifications were performed between frozen and paraffin-embedded sections of SLNs obtained from a subset of 16 MM patients ( Fig 1). As specified in the legend of Fig 1, SLNs were from patients at various stage of disease (after classification by both conventional and IHC-based histology): one AJCC stage IB, five AJCC stage IIA, four AJCC stage IIB, and six AJCC stage III cases. For PCR analysis of total RNA isolated from frozen SLNs, the first round of amplification was carried out as we previously reported,17 whereas nested primers and conditions for the second round of amplification were as described in Patients and Methods. As shown in Fig 1, RT-PCR products from frozen SLNs were separated by electrophoresis on a 2% agarose gel and results were directly visualized by ethidium bromide staining. Similar patterns of nested-PCR products with different band intensities were detected in frozen and paraffin-embedded samples from the same patients (lacking the specific amplification signals in only two archival cases: 38, with tyrosinase marker, and 37, with MelanA/MART1 marker) (Fig 1). All samples were positive for GAPDH mRNA (data not shown).
On the basis of this good agreement of PCR-positive results among the two types of specimens, 92 SLNs obtained from 75 MM patients were analyzed using the RT-PCR assay. Patients’ characteristics are listed in Table 1. Most patients (61 of 75, 81%) had histologically proven localized disease (AJCC stages IB, IIA, and IIB), and the remaining patients (14 of 75, 19%) had SLN involvement (AJCC stage III) as ascertained by IHC analysis (Table 1). Nine patients were excluded from the study because of RNA degradation.
Total RNA from paraffin sections of SLNs was screened using the RT-PCR assay to detect tyrosinase and MelanA/MART1 mRNAs. Figure 2 shows the results of a subset of archival SLNs tested with primers specific for each individual marker and for GAPDH gene expression, with detection of the specific semi-nested PCR-products (125 bp for tyrosinase, 128 bp for MelanA/MART1, and 160 bp for GAPDH). Simultaneously, PB samples from the same MM patients were analyzed by RT-PCR (as previously reported, and also specified in Patients and Methods), using an identical set of mRNA markers. Only specimens having the specific amplified bands after the second round of the nested-PCR were considered positive. Samples positive for GAPDH and showing no specific PCR-products using the two melanoma-associated markers were considered negative.
Results of the RT-PCR analysis on both PB and SLN samples from the 75 melanoma patients are listed in Table 2. Tyrosinase was expressed in 34 SLNs (45%) and 20 PB samples (27%) taken from MM patients after diagnosis; no differences in PCR-positive rates between SLN and circulation was found for the MelanA/MART1 marker (Table 2). Each marker was significantly correlated with disease stage (significance of linearity, P, was less than .01 for all markers in both PB and SLN samples, except for MelanA/MART1 in PB with P = .013). Considering the total number of PCR-positive markers, the most statistically significant correlation with disease stage was demonstrated for patients whose samples were positive for both markers in SLN (P = .006; Table 2). Thirty (49%) of 61 histopathologically proven melanoma-free SLNs were positive for at least one specific mRNA marker. Surprisingly, no melanoma-associated markers were detected in total RNA from four paraffin sections (29%) of the 14 IHC-positive SLNs used as positive controls (Table 2).
No statistical correlation between the RT-PCR results and Breslow thickness, Clark level of invasion, primary site, sex, or age was observed. Univariate analysis showed a significant correlation (P < .001) between disease stage and presence of progressive disease (PD), confirming the predictive value of clinical stage as a prognostic factor (Table 2). As listed in Table 3, analogous significant correlation was found between the rate of recurrences and the increasing number of PCR-positive markers at both PB (P = .034) and SLN (P = .001) levels. Additionally, we compared the median disease-free survival (DFS) and the overall survival (OS) with the total number of PCR-positive markers in both PB and SLN ( Tables 4 and 5). Whereas no significant difference in DFS and OS was registered for MM patients with or without melanoma-associated markers in PB (Table 4), the presence of both mRNA markers in SLN correlated with a poor clinical outcome (DFS, P < .0001; OS, P = .0047) ( Table 5).
Logistic regression multivariate analysis was performed on the totality of patients to estimate relative risk of clinical stage, presence in both PB and SLN of individual mRNA markers or total number of PCR-positive markers, as well as to adjust potential confounding effects, and to assess possible multiplicative interactions. Assuming the AJCC stage as the dependent variable, the relative risk was estimated to increase 3.8 times (P = .0078) for patients whose SLNs were positive for both tyrosinase and MelanA/MART1, whereas no other parameter (each individual mRNA marker in PB and SLN samples, total number of positive markers in PB) showed a significant prognostic value. Therefore, expression of both mRNA markers in SLNs was the only statistically independent prognostic factor for tumor progression.
Metastatic potential of melanoma is considerably greater than that reported for other solid tumors. When MM is localized (AJCC stages I and II), the 5-year DFS rate ranges from 75% to 90%; when regional lymph nodes are involved (AJCC stage III), the 5-year DFS rate falls below 50%.3 Detection of occult melanoma cells in PB by RT-PCR has been proposed as a reliable molecular approach to predict clinical behavior and to assess early tumor progression in MM patients.25 A multiple-marker RT-PCR assay has been demonstrated to be more sensitive and specific than the single-marker assay in detecting circulating MM metastases.15-17 Previously, we observed that the detected level of mRNA expression of tyrosinase and MelanA/MART1 was quite low in nonmelanoma controls (rates of false-positive were 0% and 5%, respectively),17 confirming that these two markers possess the highest specificity and reliability in detection of MM micrometastases. Additionally, a statistically significant association between AJCC stage and the presence of such tumor-associated mRNAs in PB (as an expression of circulating melanoma cells) has been widely demonstrated.15-17,19 However, the clinical significance of these circulating PCR-positive markers in MM prognosis is highly debated and not completely understood.26-29 In fact, dissemination of MM cells in PB (especially when detection is performed within a few weeks after surgical excision of the primary tumor) seems insufficient to colonize distant tissues and develop metastasis (unless metastatic tumor cells are constantly present in blood circulation; in this case, selection of viable MM cells with better capacity to invade the target tissue and with higher growth potential at the distant site is facilitated).26,30 Detection of micrometastases in regional lymph nodes could represent a useful tool for more accurate staging in patients with MM, which could improve disease management and help to obtain maximal therapeutic benefit from adjuvant therapies. Compared with conventional hematoxylin and eosin staining, IHC has already improved the sensitivity of detecting occult MM cells in serial sections of lymph nodes in the lymphatic basin draining the site of the primary melanoma.4,11 Molecular analysis by a RT-PCR assay of the same serial sections could further intensify the investigation of lymph node metastases, improving the accuracy of the MM staging.12,13,18 As reported by Morton et al,4,11 the SLN(s) may predict the histopathology of the remaining lymphatic basin in approximately 98% of cases. Therefore, RT-PCR analysis of SLN could represent a good compromise between accuracy of metastasis detection and problematic efforts of assessing the entire draining lymphatic basin, avoiding extensive evaluations of all regional lymph nodes after dissection. Sensitivity and reliability of the RT-PCR analysis is mainly dependent on sample preparation, RNA extraction, and cDNA synthesis. To avoid any PCR-based artifacts, we used standardized methods and standard quality control measures for RT-PCR analysis following the indications of the European Organization for Research and Treatment of Cancer-Melanoma Cooperative Group.31 High sensitivity of an RT-PCR assay on frozen SLNs has been reported by Bostick et al.18 Because fresh or frozen tissue sections are not usually available in all cases, we developed a new protocol to amplify total RNA from paraffin-embedded SLNs of patients with melanoma. Comparison of results between frozen and paraffin sections in a subset of 16 MM cases clearly indicated that detection of RT-PCR products was consistent in the two types of samples (Fig 1). However, our assay was not able to identify four (29%) of the 14 SLNs containing melanoma cells as assessed by the IHC analysis, suggesting that sensitivity is lowered when RT-PCR is performed on paraffin-embedded specimens. Nevertheless, the use of paraffin sections could also partially explain the very low false-positive rate for tyrosinase and MelanA/MART1 markers among the 18 tumor-free lymph nodes from nonmelanoma cancer patients analyzed, as compared with recently reported data32 (although this issue is still highly debated33). Also taking into account this weakness of our methodology, significant correlation with disease stage was demonstrated in SLN for either each mRNA marker used or for total number of PCR-positive markers. Approximately half (30 of 61, 49%) of the histologically proven tumor-free SLNs were positive for at least one specific melanoma-associated mRNA marker, and 12 (20%) of the 61 expressed both mRNA markers (Table 2). After a median follow-up of approximately 3 years, an increasing number of PCR-positive markers in SLNs were significantly associated with the risk of relapse in patients with no evidence of clinical disease. Nevertheless, poorer DFS and OS were registered in patients with histopathologically negative SLNs that expressed both mRNA markers (Table 5). These findings are consistent with those reported by Shivers et al,25 who performed an RT-PCR analysis on SLNs by examining tyrosinase mRNA expression alone. Finally, a comparison between the results on PB and SLN in the same series of MM patients indicated that although PCR-positive markers in PB were also significantly correlated to the disease stage and to the risk of recurrence, the expression of mRNA markers in SLNs has a more clinical predictive value (in terms of DFS and OS) than that detected in blood circulation (Tables 4 and 5). In conclusion, our data confirm that RT-PCR analysis of serial sections from archival SLNs could support conventional histopathologic methodologies (largely demonstrated to underestimate the real incidence of MM metastases) in improving detection of occult micrometastases. Although the sensitivity of the proposed approach is lowered by the use of the total RNA obtained from paraffin-embedded tissues, RT-PCR analysis of archival SLNs may be helpful for a more accurate staging of patients with melanoma. Further studies and a larger collection of MM patients are needed to better assess the clinicopathologic significance of RT-PCR analysis on SLN from melanoma patients.
APPENDIX
This work was funded by the Italian Ministry of Health, Regione Autonoma della Sardegna, and the National Research Council of Italy "Target Project on Biotechnology." We thank Dr Assunta Criscuolo for data management.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
-32P] ATP in a 20 µL final volume. Cycling conditions were the same as the first-round PCR with the exception of the primer annealing temperatures (58°C for TYR, and 56°C for MelanA). All PCR reactions were performed in a 9600 Thermal Cycler (Perkin Elmer). The specific PCR products of TYR, TYR semi-nested, MelanA/MART1, MelanA/MART1 semi-nested were 158, 125, 157, and 128 base pairs (bp), respectively. 
2 test. All significant parameters (below the .05 level) in the univariate analysis were included in a logistic regression for multivariate analysis. 
