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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Int J Cancer. Author manuscript; available in PMC 2010 June 1.
Published in final edited form as:
PMCID: PMC2879038
NIHMSID: NIHMS188774

Survivin expression by metastatic melanoma predicts poor disease outcome in patients receiving adjuvant polyvalent vaccine

Abstract

Survivin and livin are members of the inhibitor of apoptosis protein (IAP) family. We hypothesized that elevated expression levels of these 2 IAP genes in resected advanced-stage metastatic melanoma lesions would be associated with poor disease outcome in patients receiving a polyvalent therapeutic cancer vaccine (Canvaxin). A quantitative real-time RT-PCR (qRT) assay for survivin and livin genes was used to assess mRNA expression in 63 metastatic melanomas obtained during cytoreductive surgery of American Joint Committee on Cancer (AJCC) stage IV melanoma. Nineteen of 63 metastatic melanoma patients received Canvaxin pre- and postoperatively, and 37 patients received only postoperative Canvaxin. Expression of survivin and livin protein was assessed by immunohistochemistry (IHC) and then correlated with mRNA. Survivin mRNA was detected in 62 of 63 (98%) melanoma specimens ranging from 0–5.96 × 104 mRNA copies of total RNA. Lower mRNA copy levels of survivin significantly correlated with improved overall survival among the 37 patients who received Canvaxin postoperatively but not preoperatively (log-rank test, p = 0.023). Among patients with low survivin mRNA copies, those who received postoperative Canvaxin did significantly better than patients who received pre- and postoperative Canvaxin (p = 0.003). Livin mRNA was detectable in 60 of 63 (95%) metastatic melanoma specimens but had no significant prognostic utility. These studies demonstrate that lower levels of survivin in recurrent metastatic melanomas are associated with significantly improved survival in patients receiving postoperative adjuvant immunotherapy. Overall, the study indicates survivin expression in metastatic melanomas can significantly influence disease outcome and patient responses to immunotherapy.

Keywords: survivin, livin, melanoma, vaccine, RT-PCR

Malignant melanoma in general has variable responses to therapy.1 Systemic metastasis of melanoma is often associated with poor prognosis; the median survival for AJCC stage IV melanoma is only 8.1 months.2,3 Although metastatic melanoma is relatively resistant to chemotherapy or radiotherapy, cytoreductive surgery followed by adjuvant active-specific immunotherapy can lead to prolonged survival.4,5 In these studies, improved survival was afforded by the complete resection of multiple organ-site metastasis.6 However, it is inevitable that subclinical disease remains after cytoreductive surgery. Adjuvant active-specific immunotherapy may be effective if it can overcome tumor heterogeneity and the inherent resistance of neoplastic clones to apoptosis.79 Although tumor cells may be immunogenic and may be recognized by immune effector cells and/or antibodies, if the tumor cells are resistant to apoptosis the effect of the immune response will be limited.8 This is the same barrier faced often with chemotherapy.9

Cell death is regulated by anti-apoptotic proteins such as the Bcl-2 family and the inhibitors of apoptosis protein (IAP) family.1012 Bcl-2 inhibits caspase activity and prevents cytochrome c release from mitochondria by binding to the apoptotic protease activating factor-1 (APAF-1).10,11 The IAP survivin is located on chromosome 17q25 and encodes a 16.5 kDa protein.11,13 Although the mechanism by which survivin inhibits apoptosis has not been conclusively defined, survivin is known to block the intrinsic mitochondrial and apoptosis inducing factor (AIF)-dependent apoptotic pathway.11,14 Survivin binds to pro-caspase-9 in association with a cofactor and selectively suppresses the mitochondria/cytochrome c apoptosis pathway.15 Survivin can also bind the effector cell death proteases caspases-3 and -7 and inhibit caspase activity and cell death.16 Overexpressed survivin associates with microtubules of the mitotic spindles and has oncogenic properties by overriding the G2-M phase checkpoint.11,17 Expression of survivin is minimal or absent in adult normal cells but high in various human cancers including melanoma.11,13,18 Pennati et al.19,20 reported that ribozyme-mediated inhibition of survivin expression induced chemo-/radio-sensitivity and apoptosis in human melanoma cells.

Livin, located on chromosome 20q13, is another important IAP.21 Like other IAP members, livin can inhibit apoptosis by preventing the activation of caspase-9 by APAF-1 and cytochrome c.21 Melanoma cell lines have been reported to express livin mRNA.22,23 Although survivin and livin may block apoptosis of melanoma cells in primary and metastatic sites, the functional and target differences between these 2 proteins remain unclear. To date, the clinicopathologic significance and the expression levels of apoptosis-related proteins during progression of melanoma have not been well defined. Our hypothesis is that the level of IAP genes expressed in metastatic melanoma can be used as a surrogate of overall disease outcome in patients with advanced disease.

We have investigated the activity of Canvaxin, a polyvalent specific-active immunotherapeutic (CancerVax, Carlsbad, CA), in phase I/II trials in patients with AJCC stage III and IV melanoma at John Wayne Cancer Institute.6,24 Canvaxin consists of irradiated whole melanoma cells from 3 allogeneic melanoma cell lines that induces both cellular and humoral immune responses to multiple immunogenic melanoma-associated antigens.4,6,24 Our previous studies have demonstrated that these responses correlated with improved survival of stage III/IV melanoma patients.6,24,25 Currently, 1 FDA-approved international multicenter phase III randomized clinical trial is examining postoperative Canvaxin immunotherapy for patients who have undergone complete surgical resection for AJCC stage III melanoma.

Patients with AJCC stage IV melanoma are a heterogeneous population with variable outcomes. In phase II trials of patients matched by important prognostic factors, Canvaxin significantly prolonged median survival.6 However, the response to cytoreductive therapy with adjuvant immunotherapy is not uniform; some patients respond better than others and have prolonged survival. We hypothesized that enhanced levels of survivin and livin in metastatic melanomas would inhibit apoptosis, thereby facilitating aggressive tumor progression and resistance to therapeutic intervention. In our study, we used a quantitative real-time RT-PCR (qRT) assay to analyze survivin and livin mRNA copy levels in surgically resected metastatic melanoma specimens. MRNA copy levels in metastatic tumors were correlated to duration of survival after surgical resection of AJCC stage IV melanoma patients.

Material and methods

Patients and specimens

Tumor specimens were obtained in consultation with the surgeon and pathologist at the John Wayne Cancer Institute (JWCI). Informed human subject IRB consent was obtained from patients for the use of all specimens. All patients had stage IV malignant melanoma as defined by the current AJCC criteria.3 Pathology-verified metastatic lesions from 63 melanoma patients undergoing elective cytoreductive surgeries at Saint John’s Health Center were used. Tumor specimens were verified for minimal or no lymphocyte infiltrates. Patients were selected by the database coordinator based on Canvaxin treatment, long-term follow-up, and availability of frozen metastatic tumors from recurrent distant disease. Patients’ clinicopathologic background is shown in Table I. All 63 patients underwent complete resection of distant metastases. Thirty-seven patients had postoperative adjuvant active-specific immunotherapy (Canvaxin) in a phase II clinical trial.6 Nineteen patients had pre- and postoperative vaccine therapy in a phase II clinical trial, and 7 patients had no vaccine therapy. Regular clinical diagnostic examinations were performed in the outpatient clinic at JWCI. Tissue specimens obtained from surgery were immediately processed for RNA or were cryopreserved at −80°C until processed at a later date as previously described.26,27 Tumor specimens were coded and assessed in a blinded manner. Representative histopathology sections of melanomas were evaluated independently to determine uniformity of tumor cell content and normal cell infiltrates. The individual performing the qRT assay did not know the disease outcome. The JWCI melanoma computer database record of patients’ follow-up and history was independently provided by the database coordinator to the biostatistician (D.E.) for assessment.

TABLE I
PATIENT CLINICOPATHOLOGY CHARACTERISTICS

Melanoma cell lines

Twelve melanoma cell lines established from metastatic tumors and characterized at the John Wayne Cancer Institute (JWCI) were assessed: MA, MB, MC, MD, ME, MF, MG, MH, MI, MJ, MK and ML. All established cell lines were grown in RPMI-1640 medium supplemented with 100 ml/L heat-inactivated fetal bovine serum, penicillin and streptomycin (GIBCO, Grand Island, NY) in T75 cm2 flask as previously described.27 Total RNA was extracted from cells when cell cultures reached 70–80% confluence.

RNA isolation

Total cellular RNA from cell lines and tissue specimens was extracted, isolated and purified using Tri-Reagent (Molecular Research Center, Cincinnati, OH) as previously described.26,27 All RNA extractions were performed in a designated sterile laminar flow hood using RNAse-free labware. RNA was quantified and assessed for purity by ultraviolet spectrophotometry and RIBO-Green detection assay (Molecular Probes). Tissue processing, RNA extraction, RT-PCR assay set-up and post-RT-PCR product analysis were performed in separate designated assay rooms to prevent cross-contamination.26

Primers and probes

Primer and probe sequences were designed for the real-time quantitative PCR assay using Oligo Primer Analysis Software, version 5.0 (National Biomedical Systems, Plymouth, MN). To avoid possible amplification of contaminating genomic DNA, primers were designed so that each PCR product covered at least one intron. Fluorescence resonance energy transfer (FRET) probe sequences were as follows: survivin, 5′-TEX RED-CCGGAGCGGATGGCCGAGGCT GGC-BHQ-2-3′; livin, 5′-FAM-TGAGCTGCCCACACCCAGGAGAG-BHQ-1-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-FAM-CAGCAATGCCTCCTGCACCACCAA-BHQ-1-3′. GAPDH was used as an internal reference housekeeping-gene for all specimen mRNA, as previously described.28 All specimens were determined to have sufficiently high-integrity mRNA for PCR studies.

qRT assay

All reverse-transcriptase reactions were performed using Maloney marine leukemia virus reverse transcriptase (Protégé, Madison, WI) with oligo-dT (GeneLink, Hawthorne, NY) priming as previously described.2628 The quantitative real-time (qRT) PCR assay was performed with iCycler iQ RealTime Thermocycler Detection System (Bio-Rad Laboratories, Hercules, CA) using cDNA from 250 ng of total RNA for each reaction as previously described.28,29 Samples were amplified with a precycling hold at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min for GAPDH and survivin (annealing at 58°C for livin) and extension at 72°C for 1 min.

A standard curve for quantifying mRNA copy number was established by amplifying 9 aliquots of templates with known copy numbers (100 to 108 copies).28,29 cDNA was synthesized by RT-PCR, run on 2% agarose gel electrophoresis, and then extracted using the QIAquick gel extraction method (Qiagen, Valencia, CA). The cDNA was ligated into the pCR II-TOPO cloning vector (Invitrogen, San Diego, CA), the cDNA clones were transformed into Escherichia coli DH5-α cells, and cultures were expanded as previously described.28,29 Plasmids containing the target gene were purified and quantified for use in the qRT setup. To confirm the correct size of the inserted PCR product, plasmids were digested with specific restriction enzymes, and the cDNA clone PCR products were then run on gel electrophoresis.

The PCR amplification of the serially diluted cDNA standard templates of each marker showed a logarithmic signal increase.28,29 The standard curve was generated by using the threshold cycle (Ct) of templates in known numbers of copies. The Ct of each sample was plotted on the standard curve, and the mRNA copy number was calculated by the iCycler iQ RealTime Detection System Software (Bio-Rad Laboratories). Positive controls (melanoma cell lines) for survivin and livin, 5 standard normal controls (peripheral blood lymphocytes from healthy donors) and reagent controls (reagent without RNA or cDNA) for the qRT assays were included in each assay run. Lymphocytes were processed and RNA prepared as previously described.27 Each assay was performed at least twice to verify the results, and the mean copy number was used for analysis.

Immunohistochemistry

Expression of survivin in metastatic melanoma was assessed by IHC. Specimens were fixed in 10% formalin and paraffin-embedded by conventional pathology. Five-micron cut sections were deparaffinized in xylene, and the slides were bathed in Target Retrieval Solution (DAKO, Carpinteria, CA) at 95°C for 20 min. The sections were incubated with polyclonal goat anti-human survivin Ab (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500 and kept at 4°C overnight or rabbit anti-human livin polyclonal IgG antibody (1:100; Alpha Diagnostic International, San Antonio, TX) at 4°C overnight. Pathologically verified paraffin-embedded colorectal carcinoma tissue sections known to have survivin and livin expression were used as positive controls.22,30 Negative control slides were treated with nonimmunized goat or rabbit IgG under equivalent conditions and with no primary antibody. For the secondary developing reagents, a labeled streptavidin-biotin kit (DAKO) was used. Slides were developed with diaminobenzaminidine and counterstained with hematoxylin. The specimens were evaluated in a blinded fashion without prior knowledge of the qRT results on survivin and livin mRNA expression. The IHC results for survivin and livin were arbitrarily classified according to the intensity of immunoreactivity: (−), negative immunostaining; (±), weak immunostaining; (+), medium positive immunostaining; and (++), strongly positive immunostaining. The results of IHC and qRT for survivin or livin were compared in 12 metastatic melanoma specimens.

Statistical analysis

Patient groups based on individual factor levels were compared using Mann-Whitney U test, Kruskal-Wallis test and Spearman correlation coefficient analysis. The outcome measurement was overall survival from cytoreductive surgery for stage IV melanoma to death or last follow-up. The cumulative survival rates for patient groups were calculated using the Kaplan-Meier method and compared by using the log-rank test. Cox proportional hazards (Cox-PH) models were used for multivariate analysis of the following variables: gender, age, metastatic site, total number of metastatic sites involved and each mRNA marker expression. Backward stepwise model selection procedures were employed to find the optimal set of predictors for overall survival. All p-values that were 2-sided at a value of ≤0.05 were considered to be statistically significant.

Results

Survivin and livin mRNA levels in melanoma lines

Initially we evaluated by qRT assay survivin and livin mRNA expression in 12 established melanoma cell lines derived from metastatic melanomas (Table II). Survivin and livin were expressed in all melanoma cell lines. Data were presented as mRNA copies per 250 ng of total RNA from individual melanoma cell lines. The survivin mRNA copy number ranged from 1.73 × 104 to 1.20 × 106 copies (median 2.48 × 105 copies) and the livin mRNA copy number ranged from 1.40 × 104 to 8.18 × 105 copies (median 1.52 × 105 copies). The mean mRNA copies of survivin and livin in representative leukocytes from 5 healthy normal donors were assessed: survivin mean 27 copies/250 ng total RNA; livin mean 49 copies/250 ng total RNA. There was no significant difference amongst these donor leukocytes.

TABLE II
qRT ANALYSIS OF MELANOMA CELL LINES (n = 12)

IHC analysis of survivin and livin protein expression

Immunohistochemistry was performed on respective melanoma specimens in which qRT assay was performed to determine concordance between mRNA and protein expression levels. IHC staining for survivin and livin in 12 surgically resected metastatic melanoma from a subgroup of 12 AJCC stage IV patients showed a variation in survivin and livin immunoreactivity in melanoma cells (Tables III and andIV,IV, Fig. 1). Metastatic melanoma tumors with survivin mRNA copy numbers <500 displayed negative or weak immunostaining, whereas tumors with survivin mRNA copies ≥500 demonstrated significant enhanced immunostaining. Melanomas with elevated survivin mRNA copies demonstrated a more overall intense immunostaining (Table III). Tumors with livin mRNA copy number <100 displayed poorer immunostaining than tumors with livin mRNA copies ≥100 demonstrated more enhanced immunostaining overall (Table IV). These studies indicated that analysis of quantitative levels of survivin and livin mRNA is informative and significantly representative of the respective protein marker expression analysis (Table IV). Results were used to identify qRT cutoff\ values for survivin (500 mRNA copies) and livin (100 mRNA copies) and applied for subsequent analysis.

Figure 1
Representative IHC staining for survivin and livin protein in paraffin-embedded metastatic melanoma tissues. (a) Strongly positive immunostaining for survivin (survivin mRNA 5.24 × 103 copies); (b) negative control (same specimen as a); (c) strongly ...
TABLE III
COMPARISON OF IHC ANALYSIS TO mRNA EXPRESSION OF SURVIVIN
TABLE IV
COMPARISON OF IHC ANALYSIS TO mRNA EXPRESSION OF LIVIN

mRNA levels in metastatic tumors

Survivin and livin mRNA copy levels were assessed using the qRT assay on 63 surgically resected metastatic melanomas from 63 AJCC stage IV patients (Table V). Survivin mRNA was detected in 62 of 63 (98%) melanomas with copies ranging from 0–5.96 × 104 mRNA copies (median 1,340) per 250 ng of total RNA. Twenty-three (37%) specimens demonstrated survivin with <500 mRNA copies. Livin mRNA was detectable in 60 of 63 (95%) metastatic melanoma specimens. The livin mRNA copy number ranged from 0–9.99 × 105 copies (median 13,050) per 250 ng of total RNA from melanomas. Twelve (19%) specimens had livin mRNA with <100 mRNA copies. All melanoma tumors were positive for GAPDH mRNA, thus showing high integrity of the mRNA. There was no significant correlation between survivin and livin mRNA expression (r = 0.379; Spearman).

TABLE V
qRT ANALYSIS OF METASTATIC MELANOMA TUMORS (n = 63)

There was no significant correlation between clinicopathology factors (Table I) and survivin or livin mRNA copy number of tumors from the 63 patients. For survivin mRNA, the postoperative survival rate was higher for 23 patients with low expression (<500 copies) than for 40 patients with high expression (≥500 copies; median survival, 19.2 vs. 10.3 months), but this difference did not reach statistical significance (p = 0.10, log-rank test). Among 40 patients whose metastatic melanomas expressed higher survivin mRNA (≥500 copies), those patients who received adjuvant Canvaxin had a significantly longer survival compared to patients (n = 5) with no Canvaxin therapy (p = 0.024) (data not shown).

However, among the 37 patients who had postoperative but not preoperative Canvaxin therapy assessed, survivin mRNA copy level was significantly lower in those patients with good postoperative survival (Fig. 2a). Assessing survivin mRNA, the postoperative survival rate was significantly higher for 15 patients with low expression (<500 copies) than for 22 patients with high expression (≥500 copies; median survival, 26.1 vs. 11.3 months; p = 0.023; Fig. 2a). Moreover, when the survivin mRNA copy level was normalized to GAPDH expression level among the 37 patients, the survivin: GAPDH mRNA copy level ratio varied from 0–0.016 (mean ratio; 0.001 ± 0.003). This mean ratio (1.0 × 10−3) was used as a cutoff value for survival analysis. The survival rate was significantly higher for 22 patients with low survivin mRNA expression (survivin/GAPDH copy ratio < 1.0 × 10−3) than for 15 patients with higher survivin mRNA expression (survivin/GAPDH copy ratio ≥ 1.0 × 10−3; median survival, 24.4 vs. 11.4 months; p = 0.03; Fig. 2b). Among patients with low expression of survivin (<500 copies) who received postoperative Canvaxin vs. those who received pre- and postoperative Canvaxin, patients who received postoperative Canvaxin did significantly better in overall survival (p = 0.003; Fig. 3).

Figure 2
Correlation of survivin mRNA copies to overall survival in patients receiving only postoperative vaccine (n = 37). (a) Survivin mRNA copy number < 500 (n = 15) vs. ≥ 500 (n = 22). (b) Survivin/GAPDH copy ratio < 1.0 × 10 ...
Figure 3
Analysis of patients with survivin mRNA copy number < 500 (n = 21). Patients who received postoperative vaccine (n = 15) had significant prolonged survival than those who received both pre-and postoperative vaccine (n = 6).

Livin mRNA expression level or livin/GAPDH copy ratio did not significantly correlate with postoperative Canvaxin alone survival (Fig. 4) or pre- and postoperative Canvaxin survival (data not shown). There was no significant correlation of livin mRNA expression with any prognostic factors or disease outcome in any of the treatment groups.

Figure 4
Correlation of livin mRNA copies to overall survival in patients receiving only postoperative vaccine (n = 37). Livin mRNA copy number < 100 (n = 7) vs. ≥ 100 (n = 30).

A multivariable Cox-PH model was used to evaluate the correlation of clinicopathologic factors, molecular markers and post-operative survival. Backward stepwise model selection removed all factors from the prognostic models except for survivin mRNA expression. The survivin mRNA expression (≥500 copies) was significantly associated with a poor disease outcome (RR = 2.26; 95% CI 1.04–4.89; p = 0.04). The survivin/GAPDH ratio also yielded a relative risk of 2.11 (95% CI 1.01–4.44; p = 0.05).

Discussion

In our study, mRNA expression levels of 2 IAPs, survivin and livin, were assessed by a qRT assay in melanoma cell lines and metastatic melanomas. Results were correlated to postoperative survival of patients treated for AJCC stage IV melanoma. Because survivin and livin influence the anti-apoptotic potential and survival of melanoma cells, we hypothesized that their expression levels in metastatic melanoma may correlate with poorer disease outcome.

Expression of survivin mRNA and protein in malignant melanoma has been reported with relatively high frequencies. Grossman et al.18 reported that survivin was expressed in 15 (100%) of 15 metastatic melanomas by IHC. Survivin mRNA expression was positive in 26 (72%) of 36 sentinel lymph node metastases assessed by RT-PCR, and survivin mRNA expression was correlated with survivin protein expression assessed by IHC.31 Our study is the first to our knowledge to assess survivin mRNA copy level of metastatic melanoma specimens by a qRT assay and correlate survivin levels to disease outcome. Survivin mRNA was detected, using the optimal conditions, in 100% of metastatic melanoma cell lines and 98% of metastatic melanoma tumor specimens. As expected, the survivin mRNA copy number was heterogeneous among metastatic melanoma specimens. Among 37 patients who received postoperative Canvaxin therapy, those with <500 survivin mRNA copies in their metastatic melanoma had a significantly prolonged survival by univariate and multivariate analyses. The analysis using the ratio (survivin/GAPDH) was similar to that of survivin mRNA copies. The 2 approaches of presenting the quantitative mRNA levels were concordant. Our results also demonstrated that survivin mRNA copy number detected by a qRT assay in general correlated well with survivin protein expression assessed by IHC. These findings are consistent with studies reporting a correlation between IHC or RT-PCR analysis evidence of survivin expression and poor prognosis of patients with breast cancer, colorectal cancer, esophageal cancer and glioma.3235

Interestingly, among patients with low levels of survivin, those who received preoperative Canvaxin and developed recurrence did more poorly than patients who had only postoperative adjuvant Canvaxin alone. However, there was no statistically significant difference in disease outcome between patients with high copies of survivin receiving either pre- and postoperative vs. postoperative Canvaxin (data not shown). The sample size is small due to the limited available patients with advanced resected disease and postoperative Canvaxin. However, the trend is that patients who failed Canvaxin treatment with recurrent disease have a more aggressive type of disease whereby inherent tumor cell mechanisms may be prevalent for resistance to immune effector responses. Although clinically evident recurrent metastasis was resected, it is evident that aggressive subclinical disease remains that is unresponsive to immunotherapy. Patients with low copies of survivin receiving Canvaxin postoperatively may be more responsive to active-specific immunotherapy-induced effector responses. These studies demonstrate that recurrent disease is significantly different in patients who have and have not received Canvaxin therapy. Further studies are needed using a larger sample size to characterize this significant difference.

Our study suggests that advanced-stage melanomas with higher survivin expression may be more resistant to immune effector responses and therapeutic agents that function through the apoptotic pathway. The failure of immunotherapy against systemic metastatic melanoma can be attributed to poor recognition of target antigen(s), immunoregulation and/or poor efficacy of the immunotherapeutic. Our findings indicate that anti-apoptotic cellular mechanisms of tumor cells may also be significant factors for resistance to immunotherapy. Recently, a study demonstrated that introduction of antisense survivin into lymphoma cells significantly enhanced antitumor cytotoxic T-lymphocyte activity, apoptotic index of tumor cells and immunotherapeutic effect in vivo.36 Hausladen et al.37 reported that low urine survivin level significantly correlated with induction of remission in transitional cell carcinoma by Bacillus Calmette-Guerin (BCG) immunotherapy. In addition to its potential as a marker of chemo-/radio-sensitivity,19,20 survivin mRNA expression level holds promise as a predictive surrogate for the efficiency of active-specific immunotherapy.

A recent melanoma study demonstrated that survivin inhibits nuclear translocation of mitochondrial apoptosis-inducing factor, known to induce mitochondrial cytochrome c release and subsequent caspase activation as well as caspase-independent DNA fragmentation.38 Survivin may be a key anti-apoptosis protein, which protects both caspase-dependent and -independent apoptosis pathways. Survivin also accelerates the cell cycle by enforcing mitosis.11,17 Melanoma cells with high survivin expression thus may have more aggressive proliferation as well as resistance to apoptosis-related death.18 This aggressive phenotype might override the host immune attack and impair the efficacy of active-specific immunotherapy. This could explain why higher survivin expression levels correlated with poorer prognosis of patients with stage IV melanoma in our study. Survivin expression may be a valuable surrogate indicator of disease outcome and treatment resistance in advanced-stage patients.

Recent investigations identified survivin as a universal tumor antigen that may serve as a widely applicable target for anticancer immunotherapy.39,40 This is somewhat a paradox when survivin expression is evaluated as a surrogate of malignant potentials of melanoma as in our study. Interestingly, our study showed that Canvaxin may prolong postoperative survival in the survivin-positive (≥500 mRNA copies) group; a patient in the survivin-positive (≥500 mRNA copies) group survived more than 5 years. The 3 melanoma cell lines of Canvaxin all express survivin mRNA. Even if tumor cells that express survivin are recognized by autologous T-cells after vaccine treatment, cytotoxic T-cell-mediated apoptosis of tumor cells may be blocked by overexpression of survivin.40 Many other factors such as reduction of melanoma-associated antigen expression in melanoma cells can influence tumor escape from host immunity.1,28 However, the long-term survivor in the survivin-positive group may be related to antisurvivin immune responses. In melanoma patients, cytotoxic T-cells and antibody responses to survivin protein have been demonstrated.3941 Further investigation will be needed to explore the mechanism and efficacy of antisurvivin immune responses.

In our study, livin mRNA was highly expressed in melanoma cell lines and metastatic melanoma. Livin mRNA copy number detected by a qRT assay correlated with livin protein expression assessed by IHC. However, our results did not indicate any significant correlation between high livin mRNA expression and survival of stage IV melanoma patients who received postoperative vaccine therapy. Livin is known to have 2 transcript variants, livin α and livin β.22 Livin α and β have functional and tissue distributional differences. High levels of both livin α and β transcripts were detected in melanoma cell lines.22 In our study, the PCR primer pair designed for livin mRNA expression enables the detection of both variants. The function of each variant has not yet been defined.22 A recent study demonstrated that livin is a target of anti-tumor immunity in metastatic melanoma patients.41 The study showed that lymphocyte infiltrates in necrotic melanoma metastases included CD4+ and CD8+ T cells specific for livin; melanoma progression was associated with the loss of livin expression by IHC and the absence of lymphocyte infiltrates. The vaccine used in our study contains melanoma cell lines that express livin mRNA.

Bowen et al.23 have reported that survivin protein assessed by Western blot analysis was expressed in melanoma cell lines but not in melanocytes or keratinocytes. In contrast, livin was expressed in melanoma cells, melanocytes and keratinocytes.23 These differences suggest that each IAP member may have different functions in the regulation of apoptosis or other cellular events. The interactions if any between survivin and livin mRNA expression have not yet been defined in tumors. Survivin affects the anti-apoptotic and oncogenic behavior of melanoma cells and may have a more important impact on the survival of melanoma cells exposed to host immune effector responses generated by immunotherapy. Further studies of the biologic behavior of melanoma cells that express survivin may confirm this IAP as a potential surrogate molecular marker of metastatic melanoma progression.

Acknowledgments

We thank Ms. G. Berry’s editorial assistance and the staff of JWCI clinic. The authors did not receive any funds from a commercial sponsor for this trial or laboratory study. D.S.B.H. and D.L.M. have declared an affiliation and financial interest in CancerVax Corp. at the present time.

Grant sponsor: NIH/NCI; Grant numbers: PO1 CA 29605, CA 12528.

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