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ORIGINAL ARTICLES |
From the Department of Experimental Medicine and Pathology (AG, IS, PG, IN, GZ, LF, AMA), Division of Plastic Surgery (DR, EC, NS), University of the Study of Rome "La Sapienza," Rome, Italy; and Neuromed (Mediterranean Neurologic Institute), Pozzilli, IS, Italy (LF).
Correspondence: Address correspondence and reprint requests to: Anna Maria Aglianò, PhD, Dipartimento di Medicina Sperimentale e Patologia, Viale Regina Elena, 324-00161, Rome, Italy; Fax: 396-445-4820; E-mail: annamaria.agliano{at}uniroma1.it
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ABSTRACT |
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Methods: In this study, 129 SLNs were collected and analyzed by RT-PCR for tyrosinase and melanoma inhibitory activity (MIA) messenger RNA (mRNA) expression. Results from PCR analysis were then compared with those obtained by hematoxylin and eosin and immunohistochemistry and related to progression of disease.
Results: MIA gene expression was positive by RT-PCR in 27% of the tyrosinase-positive SLNs. When the correlation between tyrosinase and/or MIA mRNA expression and disease-free survival was evaluated by the Kaplan-Meier exact test, there was a statistically significant correlation between simultaneous tyrosinase and MIA gene expression in SLNs and progression of disease.
Conclusions: RT-PCR analysis for both MIA and tyrosinase mRNA may identify a subset of melanoma patients with a worse prognosis whom the routine methods, such as histology and immunohistochemistry, fail to identify because of the poor sensitivity of these methods.
Key Words: Melanoma • MIA • RT-PCR • Sentinel lymph node • Tyrosinase
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INTRODUCTION |
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The correlation between MART-1 mRNA negative expression in SLNs and overall survival has already been shown.9 Previously Hochberg et al.10 showed, in a small number of patients, a correlation between expression of tyrosinase, MIA, and MART-1 in the SLNs of patients with malignant melanoma and progression of disease. To verify whether a correlation between positive expression of tyrosinase and MIA markers and progression of disease does exist, we tested a larger number of SLNs in which an experienced pathologist did not find any melanocytic nevus cells by histology.
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MATERIALS AND METHODS |
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Lymphatic Mapping and SLN Biopsies
Lymphatic mapping and SLN biopsies were successfully performed at the University of Rome "La Sapienza," Department of Plastic and Reconstructive Surgery. Before surgery, cutaneous lymphoscintigraphy was performed to identify 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-headed gamma camera (Diacam; Siemens, 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 with a blue marker. Additionally, lymphatic mapping was performed during surgery with a handheld gamma probe (Neoprobe Neo-2000; Neoprobe Corporation, Dublin, OH) to accurately localize the area of highest radioactive signal intensity. The radiolabeled lymph nodes were excised and termed to represent SLNs. The lymphatic bed was then scanned until no residual radioactivity was measured. Sometimes dissection of adjacent non-SLNs of the respective regional nodal basin was performed. 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 evaluations. One half of each lymph node was examined by hematoxylin and eosin and immunohistochemistry (IHC), and the other half was used for two RT-PCR molecular assays. Thirty to forty-five 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 hematoxylin and eosin, and IHC was performed by using antibodies against HMB-45 antigen and S-100 protein (Dakopatts, Hamburg, Germany) that were detected with the avidin-biotin-peroxidase technique. Negative controls were obtained if normal animal serum was used instead of specific primary antibodies.
Reverse Transcriptase-Polymerase Chain Reaction
One microgram of total RNA extracted from one half of the frozen section of SLN tissues with the guanidium isothiocyanate method12 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 (Life Technologies, Pasleyork, UK) according to the manufacturer’s guidelines. Then 3 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 (Life Technologies) in a final volume of 50 µL; the amplification product was 516 base pairs. Three microliters of complementary DNA was amplified, and the tyrosinase primers were HTYR1 (5'-TTGGCAGATTGTCTGTAGCC-3'; location 774–793) and HTYR2 (5'-AGGCATTGTGCATGCTGCTT-3'; location 1037–1056), generating a 284–base pair 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 base pairs. The primers used to detect the MIA expression were MIA-u (5'-GTGGTCCTATGCCCAAGCTG-3'; location 1457–1476) and MIA-d (5'-GC TCACTGGCAGTAGAAATC-3'; location 3246–3265).
Amplifications were performed on a Techne Progene amplifier (Cambridge, UK). A cycle profile consisted of 30 seconds at 94°C for denaturing, 30 seconds at 60°C for annealing, and 30 seconds at 72°C for extension in amplification reactions with ß-actin and tyrosinase primers. The annealing temperature of amplification was 58°C for MIA.
All the recommended precautions were taken to avoid the possibility of false-positive results, and the preparation of the reaction mixture and 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 the M14 cell line as positive control for ß-actin, tyrosinase, and MIA.
Analysis of the RT-PCR products was performed by electrophoresis on a 2% agarose gel of 20 µL of the amplification products. Only samples that showed the specific amplification product were considered positive.
Negative Controls
A total of 40 lymph nodes from patients with nonmelanoma disease were used as negative controls for the analysis by RT-PCR of tyrosinase and MIA. Control specimens included 20 lymph node samples from patients with breast carcinoma, 10 lymph nodes from patients with colon carcinoma, and 10 lymph nodes from patients with chronic unspecified lymphadenitis.
Follow-Up
For follow-up evaluation, patients were examined prospectively for recurrence of metastatic disease at 3-month intervals. The evaluation consisted of a physical examination and routine blood investigations during a median follow-up of 43.9 months. An ultrasound examination of the regional lymph node basins and the abdomen and a chest x-ray was performed at least once a year.
Statistical Analysis
Statistical analysis was performed with BMDP statistical software, version 7 (Statistical Solutions, Saugus, MA). To evaluate the correlation between tyrosinase and/or MIA expression and disease-free survival, the Kaplan-Meier exact test was applied. Differences between survival curves were tested with the log-rank test (Mantel-Cox method). P < .05 was considered statistically significant.
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RESULTS |
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Statistical Analysis
We determined the correlation between overall survival and tyrosinase and MIA mRNA expression by the Kaplan-Meier test. Analysis of survival curves showed that the simultaneous positive expression of tyrosinase and MIA mRNA identifies patients with a significantly higher probability of melanoma recurrence than those expressing only one molecular marker or those not expressing any molecular marker (P < .01; Fig. 2).
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We then compared the specificity of the two methods. The specificity of IHC (97.3%; 95% CI, 92.4%–99.1%) was higher than that of RT-PCR (92%; 95% CI, 85.4%–95.7%), but we did not find a significant statistical difference by 
2 test (P = .075).
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DISCUSSION |
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Although several attempts have been performed to identify molecular markers in melanoma lesions and in peripheral blood from melanoma patients, few studies have focused on molecular markers for the identification of micrometastases in SLNs. Among those, tyrosinase, MART-1, and, to a lesser extent, MIA were the most commonly detected in previous studies.
Before RT-PCR analysis was performed, all samples were examined for the presence of capsular nevus cells that could give false-positive results in RT-PCR assay. Five samples that revealed capsular nevus cells after accurate histological examination were immediately excluded, and the study was performed on the remaining 129 samples. Therefore, in this study, we analyzed 129 SLNs from melanoma patients (stage I and II) for the expression of tyrosinase and MIA by RT-PCR assay.
Statistical analysis performed by the Kaplan-Meier method found a significant correlation between contemporary positive expression of MIA and tyrosinase and decreased survival (P < .05); such a correlation did not emerge in the group of patients expressing MIA or tyrosinase alone or in the group of double-negative patients.
Although the specificity of RT-PCR and IHC seems comparable, as shown by statistical analysis, RT-PCR has an higher sensitivity to detect patients with a worse prognosis. This is not surprising because of the known ability of RT-PCR to detect minimal residual disease in body fluids and in other biological materials.
Our data, which confirm the high specificity of IHC, a common method in standard staging procedures, support the role of RT-PCR in the identification of a panel of molecular markers in SLNs of melanoma patients. The contemporary use of MIA and tyrosinase markers in SLNs seems useful for selecting a subset of patients affected by melanoma with a high risk of relapse. Identification of additional molecular markers may improve the sensitivity of this method in the identification of micrometastases in SLNs of melanoma patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication October 3, 2003. Accepted for publication July 26, 2004.
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