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© 2003 American Society for Clinical Oncology Survivin, bcl-2, bax, and bcl-X Gene Expression in Sentinel Lymph Nodes From Melanoma Patients
From the Department of Experimental Medicine and Pathology, and Division of Plastic Surgery, Institute of Dermatology, University of Rome "La Sapienza", Rome, Italy. Address reprint requests to Anna Maria Aglianò, PhD, Dipartimento di Medicina Sperimentale e Patologia, Viale Regina Elena, 324-00161, Rome, Italy; email: annamaria.agliano{at}uniroma1.it.
Purpose: The expression of apoptosis-related genes, such as survivin, bcl-2, bcl-X, and bax, has been evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry in sentinel lymph nodes (SLNs) from melanoma patients and then correlated to the outcome of patients. Patients and Methods: Thirty-six SLNs were examined. After RNA extraction, an RT-PCR followed by Southern blot hybridization was performed to detect survivin, bcl-2, bcl-X, and bax mRNA. bcl-2, survivin, and bax gene expression was evaluated, whenever possible, also by immunohistochemistry at the protein level. Results: We found a significant correlation (P < .005) between survivin expression and outcome of patients; in fact, 61.5% of patients expressing survivin gene progressed or died because of the disease, whereas 38.5% are currently disease-free. Among patients negative for survivin expression, 100% are disease-free after a median follow-up time of 52.9 months. We did not find a significant correlation between bcl-2, bax, and bcl-X gene expression and outcome of patients. In fact, these genes were found equally expressed in patients with disease progression and in disease-free patients. Conclusion: Our findings show a variable expression of apoptosis-related genes in SLNs of melanoma patients; more interestingly, we found that survivin expression correlates to outcome of patients in a statistically significant way, whereas the expression of other genes, such as bcl-2, bax, and bcl-X, did not seem to correlate to progression of disease. We suggest that the detection of survivin gene expression by RT-PCR in SLNs may be a useful prognostic indicator.
TO DATE, the Breslow tumor thickness and ulceration are the most powerful predictors of survival in melanoma patients with stage I and II disease.1 Nevertheless, when the patients show lymph-node involvement, the primary melanoma prognostic factors no longer contribute as survival predictors. In recent years, the technique of sentinel lymph node (SLN) biopsy has been proven the most accurate method for staging primary cutaneous melanoma patients lacking clinical evidence of metastatic disease. A recent multicenter study2 has confirmed the accuracy of SLN biopsy in predicting nodal metastases, although an initial learning curve of 30 patient cases exists. In the past few years, several studies3,4 have recognized the apoptosis process as an important component of multistep carcinogenesis. Indeed, it seems that escape from apoptosis may be a requisite event in the development of malignant neoplasms. Genes that can influence cell viability versus cell death have been described, including genes belonging to the bcl-2 family. Related genes of this large family encode proteins that regulate apoptosis both in a negative and, in some instances, positive fashion. In addition, alterations in the expression of these genes may cause aberrations in cell death and thus contribute to cancer. The bcl-2 oncogene, first described at a translocation breakpoint in B-cell lymphoma,5 has been shown to prevent apoptosis caused by a variety of physiologic, pathologic, and pharmacologic stimuli. Furthermore, in clinical correlative studies, high levels of Bcl-2 protein have been associated with poor response to therapy in at least some groups of patients with lymphoma, leukemia, and prostate cancer. Several studies6–10 have shown high levels of Bcl-2 protein in a variety of solid tumors, including prostate carcinoma, colorectal cancer, squamous cell carcinomas of the lung, breast cancer, and nasopharyngeal malignancies. On the other hand, some authors have found decreased Bcl-2 levels in melanoma compared with benign nevi, or no difference between melanoma cell lines and normal human melanocytes.11–13 bax gene product, an intracellular partner of Bcl-2, first identified by co-immunoprecipitation with Bcl-2, is a 21-kd protein with 21% homology to Bcl-2. Expression of bax does not block apoptosis; instead, it seems to inhibit Bcl-2 function, perhaps by forming Bcl-2/Bax heterodimers or by competing with other Bcl-2 targets.14 Bcl-X, another member of the Bcl-2 family, is functionally similar to Bcl-2;15 the gene product exists in two forms, bcl-XL (long), which blocks apoptosis in many systems, and the spliced short form bcl-XS, which acts as a dominant inhibitor of Bcl-2. In a study performed by Leiter et al,16 it has been demonstrated that bcl-2 and bcl-XL gene expression increases with progression of malignant melanoma, and the authors suggest that the expression of these two genes could reflect an increased malignant potential caused by an apoptosis inhibition and growth advantage for metastatic melanoma cells. Survivin, a novel inhibitor of apoptosis, structurally analogous to IAPs (inhibitors-8 apoptosis), has been recently identified.17 Deregulation of survivin may influence both epidermal homeostasis and the development of melanoma and nonmelanoma skin cancer.18,19 More recently, Sela20 has suggested the importance of survivin expression as a prognostic marker in monitoring oncologic patients. To date, although some studies have focused on the detection of micrometastases in SLNs expressing tumor markers such as tyrosinase, melanoma-inhibiting activity (MIA), and Mart-1,21,22 the expression of apoptosis-regulating genes in SLNs from melanoma patients has been poorly investigated. In this article, we evaluated, by reverse transcriptase polymerase chain reaction (RT-PCR), the expression of genes related to apoptosis, such as bcl-2, bax, bcl-X, and survivin, in 36 SLNs from subjects affected by melanoma and correlated the results with clinical outcome of patients.
Patients and Samples Thirty-eight patients (17 males and 21 females), with an age range of 35 to 82 years (mean age, 60.5 years), who were affected by malignant melanoma and observed at the Dermatologic Clinic of the University of Rome "La Sapienza" were enrolled onto this study. Informed consent was obtained from all patients.
Tumor thickness, ulceration, level of invasion, tumor type, and stage of disease (according to tumor-node-metastasis classification of the American Joint Committee on Cancer Staging System for cutaneous melanoma) were documented.23 After surgical excision, SLNs were frozen in liquid nitrogen and stored at -80°C until use. The SLNs were tested for the presence of nevus cells by histologic examination before they were included in the study; two samples were excluded when they tested positive for the presence of these cells. We analyzed the remaining 36 SLNs for the possible presence of micrometastases, searching for tumor markers such as tyrosinase and MIA by means of two RT-PCR assays. A total of 36 SLNs were analyzed. The features of patients are listed in Table 1
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, a cutaneous lymphoscintigraphy was performed to identify those nodal basins at risk for metastatic melanoma. A total dose of 100 MBq (3 mCi) technetium-99m-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, Munich, 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 using a blue pen marker. In addition, lymphatic mapping was performed during surgery with a hand-held gamma probe (Neoprobe Neo-2000; Neoprobe Corp., Dublin, OH) to localize accurately the area of highest radioactive signal intensity. The radiolabeled lymph nodes were excised and referred to as SLNs. The lymphatic bed was then scanned until no residual radioactivity was measured. Sometimes, dissection of adjacent nonsentinel nodes of the respective regional nodal basin was performed. These additional nodes without signal intensity were referred to as nonsentinel nodes. 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, and the other half was used for two RT-PCR molecular assays. 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.
RT-PCR Amplifications were performed on a Techne Progene amplificator (Cambridge, UK). 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, bcl-2, and survivin primers. The annealing temperature of amplifications was 62°C for bax and 58°C for bcl-X. All of 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 carried out 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 a positive control for ß-actin, bcl-2, bax, survivin, and bcl-X.
Analysis of the RT-PCR Products To semiquantitatively compare the amounts of bcl-2 and bax amplification products, we performed a quantification of the hybridization signals by a densitometric scanner.
Immunohistochemistry The stained sections were observed using a light microscope, and positivity was determined by cell staining. Antigens were quantified according to the following two parameters: (1) the number of positively stained cells and (2) the intensity of the staining, ranging from pale pink to dark orange.
Follow-Up
Statistical Analysis
RT-PCR An amplification reaction with ß-actin primers was performed to confirm the suitability of RNAs. All samples were found suitable for RT-PCR. Before including the SLNs in the study, we tested all of the samples for MIA and tyrosinase expression by RT-PCR assay; all samples were found positive for tyrosinase, and 23 (64%) of 36 were found positive for MIA. We then investigated survivin, bcl-2, bax, and bcl-X gene expression by RT-PCR in all samples. Agarose gel electrophoresis of RT-PCR products is shown in Fig 1  .
Survivin gene expression was found to be positive in 26 (72%) of 36 samples and negative in 28% of samples. Among the positive patients, 16 (61.5%) of 26 died or showed progression of disease, whereas 10 (38.5%) of 26 are currently disease-free. All subjects (100%) in the group of negative patients are disease-free. bcl-2 gene expression was found in 31 (86%) of 36 patients. Sixteen (52%) of 31 positive patients died, and 15 (48%) of 31 patients are disease-free. bax gene expression was found in 25 (69%) of 36 patients, whereas 11 (31%) of 36 patients did not have bax gene expression. Thirteen (65%) of 20 patients positive for both bax and survivin expression had disease progression, whereas seven (35%) of 20 are disease-free. Among the 11 patients who were negative for bax mRNA, five (45%) of 11 were also negative for survivin gene expression and are disease-free, whereas three (27%) of the 11 patients expressing survivin died and the other three patients (27%) are disease-free.
bcl-X gene expression was found in 29 (80.5%) of 36 patients, whereas seven (19%) of 36 were negative for bcl-X expression. Among these 29 positive patients, 18 showed both the bcl-XL and bcl-XS isoform; 10 subjects were found positive only for bcl-XL isoform; and only one sample was found positive for bcl-XS alone. Twelve (43%) of 28 bcl-XL–positive patients died, and 16 (57%) of 28 are disease-free. Among the patients negative for bcl-X, three (43%) of seven died, and four (57%) of seven are disease-free. The results obtained by RT-PCR assays and the dependence between gene expression and outcome of patients are listed in Tables 1
When we compared the relative amounts of amplification products from reactions performed with bcl-2 and bax primers, we observed that 26 of 36 samples showed an amount of bcl-2 amplification product larger than that of bax; 16 of those subjects (61.5%) died or are in disease progression, whereas 10 patients (38.5%) are disease-free. Among six of 36 SLNs showing bcl-2 expression levels lower than those of bax, six (100%) of six were from patients who are disease-free. When we related survivin and bcl-2/bax ratio with the outcome of patients, we found that among 20 patients positive for survivin with the expression of bcl-2 more than bax, 16 patients died because of the disease, and four are disease-free. Dependence between outcome of patients and expression of survivin alone or survivin together to the bcl-2/bax ratio is shown in Table 3 . In four patients, no expression of bax and bcl-2 was found, and those patients are all disease-free.
Immunohistochemistry Bcl-2, Bax, and survivin protein expression were evaluated by immunohistochemistry on 17 formalin-fixed, paraffin-embedded tissue sections from SLNs of melanoma patients. Bcl-2 protein expression was found in six (35%) of 17 samples. When we compared the results obtained by immunohistochemistry with those obtained by RT-PCR, we found a correlation in eight (47%) of 17 samples; among those, 25% gave a negative result and 75% gave a positive result with both methods. In the remaining nine (53%) of 17 samples, a positive result by RT-PCR and a negative result by immunohistochemistry were found. Regarding Bax, a correlation between mRNA and protein expression was found in 15 (88%) of 17 samples; 27% of specimens were found negative by both RT-PCR and immunohistochemistry, and 73% were found positive by both assays. In 12% of samples (two of 17), a positive result by RT-PCR and a negative result by immunohistochemistry were found. Finally, a correlation between survivin mRNA and protein expression was found in 13 (76%) of 17 samples; 23% were negative for both mRNA and protein expression, and 77% were positive by RT-PCR and immunohistochemistry. In seven (41%) of 17 samples, we found a correlation between the results obtained by immunohistochemistry and those obtained by RT-PCR for bcl-2, bax, and survivin. In all samples in which we failed to find a correlation between mRNA and protein expression, the results were negative for immunohistochemistry and positive for RT-PCR because of the higher sensitivity of the latter.
Furthermore, the immunohistochemical staining was carried out to identify the cell histotypes within the metastatic lymph nodes that were positive for survivin, bcl-2, and bax. All three proteins showed positive results in correspondence to the large infiltrating neoplastic cells, whereas lymphocytes were totally negative for all three proteins (Fig 2
In this study, we investigated, by RT-PCR, the expression of apoptosis-related genes in 38 SLNs from melanoma patients. The presence of capsular nevus cells that could give false-positive results in RT-PCR assay was revealed after accurate histologic examination in two samples (5%), which were immediately excluded from the study. On the remaining 36 samples, all negative for the presence of nevus cells, we performed an RT-PCR assay for MIA and tyrosinase, which have been recently found to be the most sensitive indicators of melanoma micrometastases in SLNs.22 We then investigated the 36 SLNs for the presence of bcl-2, bax, bcl-X, and survivin by RT-PCR assay; in 47% of specimens, we also performed the detection of Bcl-2, Bax, and survivin protein by immunohistochemistry. To our knowledge, this is the first report on bcl-2, bax, bcl-X, and survivin expression in SLNs from melanoma patients detected by using a highly sensitive and specific method, the RT-PCR followed by Southern blot hybridization. We found that, in SLNs, survivin gene expression is associated with disease progression or death, whereas lack of expression identifies patients who are disease-free in a median follow-up of 52.9 months. The statistical analysis performed on results obtained from survivin RT-PCR assays shows a significant correlation between survivin gene expression and outcome of patients. These results are in agreement with the literature; many studies25–27 have found that survivin expression is related to unfavorable prognosis in patients with colorectal, gastric, lung, and bladder cancer. Particularly, in melanoma, survivin has been found expressed in metastatic and invasive forms by immunohistochemistry, thus indicating that this gene may be involved in the progression of the disease.13 Collectively, these results lead us to hypothesize that therapeutic targeting of survivin may be beneficial in patients with recurrent disease. Regarding bcl-2, the role of this gene in the emergence of melanoma metastases has been already investigated.28 In this study, we failed to find any correlation between bcl-2 expression and progression of disease. This is not surprising because it has been suggested that the expression ratio between bcl-2 and bax is a better determinant of the cellular sensitivity to apoptosis than the expression of individual members.29 Nevertheless, when we analyzed the bcl-2 and bax expression ratio in SLNs, we did not find any correlation with the outcome of patients. Indeed, this finding may represent a further confirmation of the role of survivin in the progression of disease; in fact, the majority of patients (80%) showing both positivity for survivin gene expression and high levels of bcl-2 compared with bax died because of the disease. These findings suggest that bcl-2 expression or a bcl-2/bax expression ratio greater than one in SLNs is not able to identify patients at risk for disease progression; thus, neither finding can be used as a prognostic indicator. Nevertheless, the expression of survivin and bcl-2 in SLNs may provide two independent mechanisms of apoptosis inhibition leading to growth advantage for tumoral cells. bcl-X gene expression in melanoma has been poorly investigated. In our study, bcl-X expression seems not to be related to the appearance of relapses. Leiter et al16 have shown that bcl-2 and bcl-XL expression may be associated with the progression of disease; more recently, bcl-2 and bcl-XL antisense compounds have been investigated with the aim to simultaneously downregulate bcl-2 and bcl-XL expression.30 In our study, bcl-X is expressed more as the isoform bcl-XL (with anti-apoptotic function) than bcl-XS (dominant inhibitor of bcl-2). However, we did not find any correlation with follow-up data. These results are in agreement with the work of Fulda et al,31 who described bcl-XL as the prevalent form in tumor cells, and the role of bcl-XS as a tumor suppressor is still unclear. To verify the usefulness of immunohistochemistry, which is more easily applied for prognostic purposes, we looked for the existence of a correlation between mRNA and protein expression. The lack of correlation between mRNA and protein expression of bcl-2 and survivin in 53% and 24% of samples, respectively, led us to conclude that the immunohistochemistry is not so sensitive for this purpose. However, we cannot exclude the possibility that the correlation between mRNA and protein expression would be higher on a larger number of samples. For the first time, this study demonstrates the clinical relevance of survivin-based RT-PCR assay in SLNs of melanoma patients. Therefore, because melanoma cells regularly enter the first regional lymph node (SLN) during metastatic spread, we suggest that the detection of survivin in SLNs can be used as a prognostic indicator.
Supported in part by Ministero Università e Ricera Scientifica e Tecnologica and Associazione Italiana Ricerca sul Cancro.
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25. Kawasaki H, Altieri CD, Lu CD, et al: Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res 58:5071–5074, 1998 26. Lu CD, Altieri DC, Tanagawa N: Expression of a novel apoptosis gene, survivin, correlates with tumor cell apoptosis and p53 accumulation in gastric carcinoma. Cancer Res 1:1808–1812, 1998 27. Swana HS, Grossman D, Anthony JN, et al: Tumor content of the antiapoptotic molecule survivin and recurrence of bladder cancer. JAMA 341:452–453, 1999 28. Gradilone A, Greco C, Gazzaniga P, et al: BAX gene expression in melanoma metastases. J Invest Dermatol106:382, 1996 (letter)[CrossRef][Medline] 29. Gazzaniga P, Gradilone A, Vercillo R, et al: bcl-2/bax mRNA expression ratio as prognostic factor in low grade urinary bladder cancer. Int J Cancer 69:100–104, 1996[CrossRef][Medline]
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31. Fulda S, Friesen C, Los M, et al: Betulinic acid triggers CD95 (APO-1/Fas)-and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res 57:4956–4964, 1997 Submitted August 8, 2001; accepted September 18, 2002.
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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
