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Annals of Surgical Oncology 10:396-402 (2003)
© 2003 Society of Surgical Oncology

ORIGINAL ARTICLES

Prognostic Significance of Reverse Transcriptase-Polymerase Chain Reaction–Negative Sentinel Nodes in Malignant Melanoma

Diego Ribuffo, MD, Angela Gradilone, PhD, Marco Vonella, MD, Stefano Chiummariello, MD, Emanuele Cigna, MD, Nickolaos Haliassos, MD, Rita Massa, MD, Ida Silvestri, PhD, Stefano Calvieri, MD, Luigi Frati, MD, Anna Maria Aglianò, PhD and Nicolò Scuderi, MD

From the Division of Plastic Surgery (DR, SC, EC, NS), Department of Experimental Medicine and Pathology (AG, IS, LF, AMA), Institute of Histology and Embriology (NH), Service of Nuclear Medicine (RM), and Department of Dermatology (SC), "La Sapienza" University, Rome, Italy; Operative Unit of Plastic Surgery (MV), Policlinico "Mater Domini," Catanzaro, Italy; and NEUROMED (LF), Mediterranean Institute of Neurosciences, Pozzilli (IS), Italy.

Correspondence: Address correspondence and reprint requests to: Diego Ribuffo, MD, Cattedra di Chirurgia Plastica, Dipartimento di Dermatologia e Chirurgia Plastica, Viale del Policlinico, 155-00161 Rome, Italy; Fax: 39-06-491525; E-mail: diegoribuffo{at}libero.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Polymerase chain reaction (PCR) is a molecular biology technique that can detect a single metastatic cell in 106 to 107 normal cells. Its use has been proposed as an additional new method for the detection of malignant melanoma nodal metastases in the sentinel lymph node (SLN) to improve the detection rate guaranteed so far by standard histology (hematoxylin and eosin; H&E) and immunohistochemistry (IHC).

Methods: Since October 1995, 137 patients with primary cutaneous melanoma (Breslow thickness, .75–4 mm) have undergone surgery for selective lymphadenectomy. To identify the SLNs, every patient had preoperative lymphoscintigraphy and a vital dye perilesional injection, followed by a gamma probe–guided operation.

Results: In 134 patients at least one SLN was detected, with a detection rate of 98%. Every SLN was examined by H&E and IHC (S-100 antigen and HMB-45 protein). The messenger RNA codifying for tyrosinase and MART-1 (melanoma antigen recognized by T cells) was used as the target sequence for the reverse transcriptase (RT)-PCR. The results showed 11% positive SLNs with IHC and H&E examination and 63% with RT-PCR. No recurrence was noted at follow-up in the group with RT-PCR–negative nodes (absence of false-negative cases).

Conclusions: In our experience, RT-PCR SLN negativity is achieving a very favorable prognostic significance. However, RT-PCR positivity is still to be evaluated. Furthermore, results obtained with this method have been shown so far to be independent of Breslow’s tumor thickness.

Key Words: Melanoma • Sentinel lymph node • Polymerase chain reaction • Tyrosinase • MART-1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the progress in early diagnosis, the incidence of primary malignant melanoma is rapidly increasing.1 The prognosis of malignant melanoma is correlated especially with the pT category of the primary tumor, which is based on Clark’s and Breslow’s levels of invasion and tumor thickness.1,2 During recent years, additional clinical variables (anatomical location of the primary tumor, sex, and tumor characteristics [histological type, phase of tumor growth, ulceration, mitotic rate, lymphocyte infiltration, and vascular invasion]) were demonstrated to influence or reflect the metastatic potential of malignant melanoma, although these factors are not independent of Breslow’s tumor thickness.1–10 Despite all these additional factors that have improved the prognostic classification, the individual outcome of patients with primary melanoma still remains difficult to predict. It seems, though, that the involvement of regional lymph nodes draining the primary tumor site strongly relates to accurate prognostic tumor staging. In fact, the 5-year survival rate for early-stage melanoma (American Joint Committee on Cancer [AJCC] stages I and II) decreases to <50% in patients when metastases are identified in the regional lymph nodes (AJCC stage III).1,2

The sentinel lymph node (SLN) biopsy11 is based on the concept that a definite region of the skin drains specifically to an initial lymph node, which is therefore called the SLN of the afferent lymphatic system. Standard histological (hematoxylin and eosin; H&E) and immunohistochemical (IHC) analysis of the SLN might predict the presence or absence of metastatic melanoma cells.11–14 The importance of detecting tumor cells in these lymph nodes is crucial because of the specific therapeutic possibilities of adjuvant therapy and follow-up procedures, applied in an early stage, that can reduce the percentage of tumor progression or increase the survival rate, particularly in patients with low-risk melanoma. Serial sectioning with H&E and IHC by using more than one antigen as a target of HMB-45 and S-100 antibodies improves the sensitivity of melanoma cell identification. The routine use of H&E and IHC examinations has shown, during these years, a population of false negatives (i.e., patients with "negative" SLNs showing progression of disease).

The reverse transcriptase-polymerase chain reaction (RT-PCR) has been adapted for lymph node analysis in patients with cutaneous melanoma because of its major sensitivity. Tyrosinase, as the key enzyme in melanin biosynthesis, is known to be the most specific marker for cells derived from the melanocytic lineage. Therefore, detection of tyrosinase messenger RNA (mRNA) identified by RT-PCR has been proposed to be a marker for the presence of melanoma cells, although it has been criticized for upstaging the disease. Furthermore, because melanomas are heterogeneous, in the presence of tumor-associated gene expression, melanoma cells may go undetected when only a single marker for RT-PCR is used. To avoid false negatives, we used a second antigen, MART-1 (melanoma antigen recognized by T cells). This antigen is expressed in most melanoma cell lines and in all melanocyte cell lines tested. The two markers combined would permit further evaluation of the presence of tumor cells in SLNs.

Therefore, the goal of this study was to evaluate the use of two tumoral markers versus conventional IHC and H&E methods and to address the question of whether the identification of minimal residual disease by RT-PCR reliably indicates a high risk for developing melanoma recurrences. Finally, we analyzed the supposed correlation of RT-PCR positivity with Breslow’s tumor thickness to see whether RT-PCR status could be considered a further prognostic factor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
A total of 134 patients with AJCC stage I and II cutaneous melanoma (tumor thickness, .75–4 mm) with clinically negative lymph nodes (assessed by physical and staging evaluation: lymph node ultrasound, chest x-ray, abdominal ultrasound, and computed tomography) were included in this study. There were 69 men and 65 women, whose ages ranged from 21 to 81 years (median, 55.5 years). All 134 patients had cutaneous melanomas. Patients gave informed consent for all aspects of the investigation.

Lymphatic Mapping and SLN Biopsy
Lymphatic mapping and SLN biopsies were successfully performed from October 1995 to March 2001 at the University of Rome "La Sapienza," Department of Plastic and Reconstructive Surgery. Before surgery, cutaneous lymphoscintigraphy was performed to identify those nodal basins at risk for metastatic melanoma. A total dose of 100 MBq (3 mCi) of 99mTc-labeled nanocolloids was injected intracutaneously in six to eight equal parts around the primary melanoma or around the biopsy site if the melanoma had been previously excised. A single-head gamma camera (Diacam; Siemens, Munchen, Germany) was used to identify all basins at risk for metastatic disease and to localize the SLNs. The specific region of initial entrance and persistent accumulation of radiolabeled substance was assumed to represent the SLNs. Images were taken to define the anteroposterior position of the SLNs in two different views, and the SLNs were marked on the skin by using a blue marker. Additionally, lymphatic mapping was performed during surgery with a handheld gamma probe (Neo-2000; Neoprobe Corp., Dublin, OH) to localize accurately the area of highest radioactive signal intensity. The radiolabeled lymph nodes were excised and termed to represent SLNs. The lymphatic bed was then scanned for evaluation until no residual radioactivity was measured. Sometimes, dissection was performed of adjacent non-SLNs of the respective regional nodal basin. These additional nodes without signal intensity were termed non-SLNs. In patients who had melanomas in areas that drained concomitantly into two nodal drainage basins, SLN biopsy was performed in both nodal basins. SLNs were divided into two pieces for separate evaluation. One half of each lymph node was examined by H&E and IHC, and the other half was used for the two molecular analysis. Thirty to 45 minutes before surgery, the lymphatic mapping technique was supplemented by intradermal administration of 1 to 2.5 mL of isosulfan blue dye (Patent Blue V, 2.5%; SALF Spa, Bergamo, Italy) around the site of the primary melanoma.

Histology and IHC
One half of each lymph node was fixed in 5% formaldehyde and embedded in Paraplast (Monoject, St. Louis, MO). Sections were stained with H&E, and IHC was performed by using antibodies against HMB-45 antigen and S-100 protein (Dakopatts, Hamburg, Germany) and detected with the avidin-biotin-peroxidase technique. Negative controls were obtained if normal animal serum was used instead of specific primary antibodies.

Reverse Transcription-Polymerase Chain Reaction
One microgram of total RNA extracted from the frozen tissues with the guanidinium isothiocyanate method15 was reverse-transcribed in a final volume of 20 µl containing 20 mM of Tris HCl (pH 8.3), 50 mM of KCl, 2.5 mM of MgCl2, 100 pmol of random examer, and 50 U of murine leukemia virus RT (Invitrogen, Carlsbad, CA), according to the manufacturer’s guidelines. Then, 5 µl of complementary DNA was amplified in PCR buffer containing 25 pmol each of upstream (5'-GTGGGGCGCCCCAGGCACCA-3'; location 103–122) and downstream (5'-CTCCTTAATGTCACGCACGATTTC-3'; location 619–642) ß-actin primers and 1.25 U of Platinum Taq polymerase (Invitrogen) in a final volume of 50 µl; the amplification product was 516 base pairs (bp). A nested RT-PCR with specific oligonucleotide primers for human tyrosinase was performed by using 5 µl of complementary DNA; the tyrosinase primers were HTYR1 (5'-TTGGCAGATTGTCTGTAGCC-3'; location 774–793) and HTYR2 (5'-AGGCATTGTGCATGCTGCTT-3'; location 1037–1056), generating a 284 bp amplification product. For reamplification with the nested primers, .5 µl of the first round of amplification was amplified in a final volume of 50 µl; the nested primers used were HTYR3 (5'-GTCTTTATGCAATGGAACGC-3'; location 818–837) and HTYR4 (5'-GCTATCCCAGTAAGTGGACT-3'; location 1006–1025), generating an amplification of 207 bp. The primers used to detect the presence of MART-1 were the following: forward, 5'-ACTGCTCATCGGCTTTG-3', location 173–190; and reverse, 5'-TCAGCATGTCTCAGGTG-3', location 423–439; these primers generated a band of 268 bp. The nested MART-1 primers were the following: forward, 5'-GGATACAGAGCCTTGATGG-3', location 210–228; and reverse, 5'-TCTCGCTGGCTCTTAAGG-3', location 405–422, generating a 213-bp product. The forward and reverse ß-actin and tyrosinase primers used in the reactions of amplification were selected to be located in different exons as not to amplify contaminating DNA.

Amplifications were performed on a Techne Progene (Cambridge, UK) amplifier. A cycle profile consisted of 30 seconds at 94°C for denaturation, 30 seconds at 60°C for annealing, and 30 seconds at 72°C for extension in amplification reactions with ß-actin primers. The annealing temperature of amplifications was 55°C for tyrosinase. All the recommended precautions were taken to avoid the possibility of false-positive results, and the preparation of reaction mixture and the analysis of amplified products were performed in separate rooms. Each RT-PCR experiment included a sample without RNA as a negative control and RNA extracted from an M14 cell line as a positive control. Twenty microliters of the amplification products was electrophoresed on 2% agarose gels, followed by ethidium bromide staining. The amplification with ß-actin was performed to confirm the suitability of RNAs. All samples were found suitable. Only samples that showed specific bands after a second round of amplification with the nested primers were considered positive.

Negative Controls
A total of 85 lymph nodes from patients with nonmelanoma disease were used as negative controls for tyrosinase RT-PCR. Control specimens included 24 lymph node metastases from 13 patients with breast carcinoma, 32 nodal metastases from 20 patients with colon carcinoma, 18 lymph nodes from 10 patients with lymphoma, and 11 nodes with chronic unspecified lymphadenitis from 4 patients.

Follow-Up Evaluation
Patients were examined prospectively for recurrent or metastatic disease at 3-month intervals. The evaluation consisted of a physical examination and routine blood investigations. An ultrasound examination of the regional and abdominal lymph node basins and a chest x-ray were performed at least once a year. Computed tomography and magnetic resonance imaging were also performed in patients with findings suggestive of metastatic melanoma. The median follow-up period was 42 months (minimum, 12 months; maximum, 63 months).

Statistical Analysis
The 95% confidence intervals for the median of Breslow’s tumor thickness were calculated by using the exact method for small samples, on the basis of a binomial distribution.16 Tumor thickness between patient groups that were defined by the SLN status was compared by using the Mann-Whitney U-test. Disease-free interval was defined as the time from SLN biopsy to the date of last follow-up evaluation or to the date of first recurrence. Disease-free survival was calculated by using Kaplan-Meier estimates.17 Differences between survival curves were tested with log-rank statistics. To identify independent relevance, we investigated the influence of the histopathological status of the SLN, of the RT-PCR status of the SLN, and of Breslow’s tumor thickness on disease-free survival by Cox’s proportional hazards regression model.18 In all statistical analyses, P < .05 was defined to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Successful identification of the SLN was achieved in 134 (98%) of 137 patients with stage I and II cutaneous melanoma and a tumor thickness of at least .75 mm (median, 1.82 mm). In 140 of 143 nodal basins, at least one SLN was detected by simultaneously using a noninvasive gamma probe mapping technique and a vital blue dye lymphatic coloration. In these 134 patients, 287 regional lymph nodes, ranging from 1 to 4 (median, 2) per patient, were obtained and analyzed for the presence of melanoma micrometastasis. The presence of micrometastasis was evaluated by using IHC and RT-PCR for tyrosinase and MART-1.

IHC evaluation (for HMB-45 antigen and S-100 protein) of the 287 SLNs from 134 patients showed 28 lymph node metastases from 15 patients (11%). RT-PCR evaluation for tyrosinase and MART-1 identified melanoma micrometastases in 186 SLNs of 85 patients (63%; Table 1). The detection of melanoma cell micrometastasis in the SLNs was significantly increased by using tyrosinase and MART-1 RT-PCR in comparison with IHC, thus showing the high sensitivity of this assay (Table 1; P < .001; {chi}2 test). Tyrosinase mRNA and MART-1 were detected in all lymph nodes that were positive by IHC. If histopathologically negative SLNs were analyzed by RT-PCR, micrometastases were found in 59 (50%) of 119 patients. In this group of RT-PCR–positive patients, 54 (90%) of 59 were found to be tyrosinase/MART-1 positive, whereas 5 (10%) were MART-1 positive only. Because false-positive amplification of tyrosinase can be due to the presence of nodal nevi, we re-examined all slides of the lymph nodes that were negative by IHC and that yielded positive RT-PCR results. Nevus cell clusters were identified in the capsule of 6 lymph nodes from 5 of the 75 patients. The nevus cells showed positivity for S-100 protein but no expression of HMB-45 antigen. These six lymph nodes were reclassified as negative for micrometastatic disease.


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  		TABLE 1. Detection of nodal micrometastasis by immunohistochemistry and RT-PCR
 
The results were specific because from 85 lymph nodes of patients with nonmelanoma disease, 82 were found to be negative for tyrosinase and MART-1 gene expression by RT-PCR (HL60 promyelocyte cells). In the capsule of the three positive lymph nodes, after re-examination, were identified clusters of nevus cells. These results are in keeping with the literature and strongly suggest the high specificity of tyrosinase and MART-1 in melanoma cell tumors.

The overall survival rate was 86%. To evaluate the association of nodal RT-PCR status and major prognostic factors for primary melanoma (Breslow’s tumor thickness, Clark’s level of invasion, histology, age, and sex), three groups of patients were formed on the basis of the results of IHC and RT-PCR (Table 2). Group l included patients with at least one histopathologically proven metastatic lymph node in the nodal basin (median tumor thickness, 2.00 mm). Fifteen patients were included with 28 SLNs. Metastatic disease was shown by IHC in 28 SLNs. Tyrosinase/MART-1 mRNA could be detected in all 28 of these lymph nodes. Group 2 comprised 70 patients with 210 SLNs negative by IHC evaluation but positive by RT-PCR in at least 1 lymph node of the regional lymph node basin (median tumor thickness, 1.87 mm). Submicroscopic melanoma cells were identified by tyrosinase and/or MART-1 RT-PCR in 197 SLNs from these 70 patients. Group 3 consisted of 49 patients with 49 SLNs with negative results in both diagnostic techniques (median tumor thickness, 1.80 mm).


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  		TABLE 2. Patient groups defined by lymph node evaluation with IHC and RT-PCR
 
The nodal RT-PCR status was not shown to correlate with tumor thickness because RT-PCR–positive patients (groups 1 and 2; median tumor thickness, 1.92 mm) did not demonstrate a significantly higher median tumor thickness than did RT-PCR–negative patients (group 3; median tumor thickness, 1.80 mm; P < .1; Mann-Whitney U-test). Group 2 patients, especially, who were defined as having micrometastatic disease detectable by RT-PCR only, did not show a significantly higher tumor thickness than did patients with negative results obtained by both methods (group 3; P > .1; Mann-Whitney U-test; Fig. 1). Therefore, the number of lymph node micrometastases detected by tyrosinase RT-PCR does not increase with tumor thickness of primary melanoma.



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  		FIG. 1. The boxplots represent the distribution of Breslow’s tumor thickness of the three groups of patients on the basis of the results of immunohistochemistry (IHC), hematoxylin and eosin (H&E), and reverse transcriptase-polymerase chain reaction (RT-PCR). Group 1, IHC and PCR positive; group 2, IHC negative and PCR positive; group 3, IHC and PCR negative. The lower boundary of the boxes is the 25th percentile, and the upper boundary is the 75th percentile. The horizontal lines inside the box represent the median.

 
The Kaplan-Meier curves for disease-free survival were strongly related to the SLN positivity of metastasis as defined by the three groups of patients (Fig. 2). Among patients with histopathologically negative SLNs, those positive by molecular analysis had a significantly higher probability of melanoma recurrence than those negative by RT-PCR (P < .01). In addition, histopathologically positive patients had a significantly lower disease-free interval than those exclusively positive by RT-PCR (P < .001).



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  		FIG. 2. Kaplan-Meier disease-free survival rate. Probability of disease-free survival rate for immunohistochemistry (IHC)-negative and reverse transcriptase-polymerase chain reaction (RT-PCR)–negative patients (Histo neg. PCR neg.), for patients IHC negative but RT-PCR positive (Histo neg. PCR pos.), and for patients IHC and RT-PCR positive (Histo pos. PCR pos.).

 
To identify prognostic factors that influence disease-free survival, Cox hazard regression analysis was performed, considering Breslow’s tumor thickness and the histopathological or RT-PCR status of SLN. In this model, Breslow’s tumor thickness was independent from RT-PCR lymph node status in predicting disease-free survival. Furthermore, RT-PCR and IHC SLN status were the most significant prognostic factors (Table 3).


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  		TABLE 3. Multivariate analysis of factors affecting disease-free survival
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Histopathologic evaluation of SLN status is now considered to be the most important prognostic factor influencing survival in patients with stage I and II melanoma.19 The 5-year survival rate for localized melanoma (AJCC stages I and II) is approximately 80% but decreases to 50% with microscopic lymph node involvement (AJCC stage III). Accurate staging of the lymphatic basin draining the primary melanoma has become increasingly important because adjuvant therapy, which has been shown to improve survival, is now available. New adjuvant therapies for melanoma patients with lymph node metastases stress the importance of accurately staging the illness. To obtain the most accurate analysis of the SLN, the RT-PCR technique has been recently introduced over the conventional sectioning with H&E and IHC.20 The clinical significance of RT-PCR has been discussed since its introduction, because it might overestimate SLN positivities,21–23 although recent studies seem to validate it.24–26

Our experience highlights a possible new tool in malignant melanoma prognosis. The most important finding in our study is the absence of false-negative results when the SLN is examined with RT-PCR, compared with the false-negative rate obtained with IHC27 and H&E.28 Although our median follow-up is not complete, we know that most melanomas recur in 2 years’ time. Recurrence rates were significantly higher in patients with submicroscopic tumor cells identified by RT-PCR than in patients with negative results. These data well support our study and clarify the clinical significance of RT-PCR negativity as a prognostic factor for the absence of tumoral progression.

The clinical significance of RT-PCR positivity is, however, yet to be elucidated. The presence of nevus cells in tumor-draining lymph nodes might be a source for false-positive detection of tyrosinase mRNA. The occurrence of nevocytes in melanoma-draining lymph nodes has been reported to be as high as 10%.29–32 We found that 6 lymph nodes from the 174 lymph nodes that yielded a negative IHC result but a positive RT-PCR result and 3 lymph nodes from 85 negative control patients showed clusters of intracapsular nevus cells. We reclassified these six lymph nodes as negative for melanoma micrometastasis. Nodal nevi were characterized morphologically by subcapsular location and strong immunoreactivity for S-100, but not for HMB-45.

In our experience, RT-PCR positivity did not correlate with Breslow’s thickness. So far, only two studies have addressed this problem, and their findings are contradictory.33,34 Our findings show that with the increase in size of the primary tumor, the RT-PCR positivity rate did not increase proportionally. This might explain the well-known finding of patients with low-thickness melanoma having disease progression.

In conclusion, the most probable significance of RT-PCR positivity is disease spread, which will not necessarily have clinical expression. We hypothesize that analysis of SLNs by tyrosinase/MART-1 RT-PCR can greatly improve the diagnostic value of this nodal staging technique and may serve as an important prognostic factor in patients with stage I and II cutaneous melanoma. Furthermore, additional studies are needed to determine whether MART-1 is a more sensitive marker for occult disease than tyrosinase. Another important topic to be addressed is how these patients should be treated. Although there is a general consensus on lymph node dissection for H&E and IHC SLN-positive patients (which is our policy), it has been shown that this might be insufficient.35 However, we do believe that not all RT-PCR–positive patients would benefit from a lymph node dissection.


   Footnotes
 
In 134 patients studied with a 2- to 5-year follow-up, 11% of sentinel lymph nodes were positive when examined with hematoxylin and eosin and immunohistochemistry, and 63% were positive when examined with polymerase chain reaction (PCR). No recurrence was noted in the group with PCR-negative nodes.

Received for publication June 6, 2002. Accepted for publication December 4, 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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In Reply:
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