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Clinical Cancer Research Vol. 11, 1237-1246, February 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Clinical

Impact of Gene Polymorphisms on Clinical Outcome for Stage IV Melanoma Patients Treated with Biochemotherapy: An Exploratory Study

Dongxin Liu1, Steven J. O'Day3, Dongyun Yang2, Peter Boasberg3, Robert Milford4, Tim Kristedja3, Susan Groshen2 and Jeffrey Weber1

Departments of 1 Medicine and 2 Preventive Medicine, University of Southern California Keck School of Medicine, Los Angeles, California and 3 Divisions of Medical and Surgical Oncology and 4 Weil Medical Oncology Research Laboratory, John Wayne Cancer Institute at St. John's Health Center, Santa Monica, California

Requests for reprints: Jeffrey Weber, Department of Medicine, University of Southern California Keck School of Medicine/Norris Cancer Center, 1441 Eastlake Avenue, Los Angeles, CA 90033. Phone: 323-865-3360; Fax: 323-865-0061; E-mail: jweber{at}usc.edu.


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Biochemotherapy can achieve high response rates in advanced melanoma, but the factors that influence regression and survival remain unknown. The present exploratory study tested the hypothesis that cytokine gene polymorphisms predict clinical outcome in stage IV melanoma patients treated with biochemotherapy.

Experimental Design: Ninety patients with stage IV melanoma were treated with biochemotherapy, including cisplatin, vinblastine, and dacarbazine combined with interleukin (IL)-2 and IFN-{alpha} either with or without tamoxifen. Cytokine gene polymorphisms for IFN-{gamma} (+874A->T) and IL-10 (–1082G->A) were assessed. X-ray repair cross-complementing gene 1 (XRCC1; Arg399Gln), xeroderma pigmentosum complementary group D (XPD; Lys751Gln), and excision repair cross-complementing gene 1 (ERCC1; codon 118) DNA repair polymorphisms were also determined.

Results: IFN-{gamma} (+874A->T) gene polymorphism was statistically significantly associated with response (P = 0.001), progression-free survival (P = 0.0012), and overall survival (P < 0.001), whereas the IL-10 polymorphism was marginally associated with response (P = 0.03) and overall survival (P = 0.065). Multivariate analysis revealed that IFN-{gamma} (+874A->T) independently predicted overall survival (P = 0.003). The ERCC1 polymorphism was weakly associated with overall survival (P = 0.045). Combining polymorphisms for IFN-{gamma}, IL-10, and ERCC1 stratified patients into four distinct groups with significantly different clinical outcome (P < 0.001), so that patients with more "favorable" polymorphisms had a better outcome.

Conclusions: Cytokine gene polymorphisms predicted clinical outcome for advanced melanoma patients who received biochemotherapy. The combined effects of multiple genetic polymorphisms may provide more accurate prognostic information. Additional independent studies are needed to confirm these pilot findings.

Key Words: gene • DNA repair • cytokine


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metastatic melanoma poses a serious therapeutic problem because the 5-year mortality for stage IV melanoma is >95%. Various combinations of drugs to improve the outcome for stage IV melanoma have been explored. Treatment regimens that combine chemotherapy [e.g., cisplatin, vinblastine, and dacarbazine (CVD)] and biological agents [e.g., interleukin (IL)-2 and IFN-2], called biochemotherapy, have achieved response rates as high as 50% to 60% and ~12-month median survivals (1–3). However, the factors that influence which patients respond to those regimens are unknown. Clinical outcome may be influenced by genetic polymorphisms, which are natural variations of single base pairs in genomic DNA encoding genes that may influence the activity of the chemotherapy and immunologic agents included in the biochemotherapy regimen.

Biochemotherapy includes cytotoxic agents as well as cytokines that mediate tumor regression by completely different mechanisms. Therefore, in the present study, we focused on gene polymorphisms for cytokine genes (IFN-{gamma} and IL-10) and included several DNA repair genes [excision repair cross-complementing gene 1 (ERCC1), X-ray repair crosscomplementing gene 1 (XRCC1), and xeroderma pigmentosum complementary group D (XPD)] known to influence outcome after treatment with platinum.

Melanoma is an immunologically responsive or immunogenic tumor (4). This idea is supported by the responsiveness of melanoma to IL-2, IFN-{alpha}, and other cytokines (5); the fact that prognosis in primary melanoma is influenced by the level of lymphocytes infiltrating the primary tumor (6); and the known spontaneous regression of primary melanomas (7). IFN-{gamma} and IL-10 are probably the most important and widely studied cytokines in melanoma. IFN-{gamma} produced by Th1 cells increases MHC expression on epithelial and endothelial cells, augments the expression of IL-2 receptors on T cells, and increases the cytolytic activity of natural killer cells and macrophages (8). IFN-{gamma} can up-regulate HLA class II expression on lymphocytes in melanoma patients as well as on melanoma tumor cells (9, 10). IFN-{gamma} gene polymorphisms were shown to influence levels of cytokine production. The 12 CA repeat microsatellite allele at the first intron of the IFN-{gamma} gene was shown to be associated with a higher level of in vitro cytokine production (11). The same group reported an absolute correlation between the 12 CA repeat allele and the presence of the T allele at a single nucleotide polymorphism located at the +874 position (+874T->A) from the translation start site. This coincided with a putative nuclear factor-{kappa}B binding site that may be important for the induction of constitutively high IFN-{gamma} production (12).

In contrast, IL-10 is an anti-inflammatory cytokine that drives Th2 helper T-cell polarization and inhibits the production of IL-1, IL-2, tumor necrosis factor, IFN-{gamma}, and other Th1 cytokines (13, 14. IL-10 has been detected in the serum at high levels in many cancers, including melanoma (15, 16). High IL-10 levels have been associated with poor survival in melanoma and other malignancies (17). The IL-10 gene promoter is polymorphic, and IL-10 promoter alleles at nucleotide –1082 are associated with differing levels of IL-10 production (18).

DNA repair capacity in tumor cells is believed to determine resistance to chemotherapy (19). More efficient DNA repair would lead to increased resistance to chemotherapeutic agents that act by DNA cross-linking or dimer formation, particularly alkylating agents. Because alkylating agents cisplatin and dacarbazine are part of the biochemotherapy regimen, we reason that DNA repair polymorphisms shown to impact on the treatment of other cancers (e.g., colorectal cancer) with those drugs might also influence therapeutic outcome in melanoma (20–23).

In humans, there are five major DNA repair pathways: direct repair, base excision repair, nucleotide excision repair, mismatch repair, and double-strand break repair (24). ERCC1 is an essential member of the nucleotide excision repair pathway (25, 26), and a single nucleotide polymorphism at codon 118 (C->T) is reported to be associated with altered ERCC1 mRNA levels (27) and clinical outcome in advanced colorectal cancer patients treated with platinum-based chemotherapy (21). The polymorphism of the XPD gene at codon 751 (Lys751Gln) involved in the nucleotide excision repair pathway and polymorphism of the XRCC1 gene at codon (Arg399Gln) involved in base excision repair pathway are also shown to be correlated with DNA repair capacity (28–31).

We hypothesized that cytokine gene polymorphisms individually and/or in combination may affect clinical outcome for advanced melanoma patients who receive biochemotherapy. To test this hypothesis, we conducted an exploratory study that included 90 stage IV melanoma patients, all of whom received CVD as well as IL-2 and IFN-{alpha}. The presence of cytokine gene polymorphisms [IFN-{gamma} (+874T->A) and IL-10 (–1082G->A)] as well as DNA repair gene polymorphisms [ERCC1 (codon 118), XRCC1 (Arg399Gln), and XPD (Lys751Gln) were determined, and their associations with clinical outcome (objective response, progression-free survival, and overall survival) were examined.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients. Ninety patients with metastatic melanoma (American Joint Committee on Cancer stage IV) received concurrent biochemotherapy. Patients were treated on two different prospective clinical trials approved by the Institutional Review Board of St. John's Hospital (Santa Monica, CA). Eligibility was based on the following criteria: age between 18 and 70 years, histologically confirmed diagnosis of malignant melanoma with measurable American Joint Committee on Cancer stage IV disease, Eastern Cooperative Oncology Group performance status of 0 to 2, and acceptable organ function (total bilirubin <2.0 mg/dL, serum creatinine <1.6 mg/dL, WBC count >3,000/µL, and platelet count >100,000/µL). Before treatment, blood was drawn and subsequently used to determine gene polymorphisms for all patients. All patients gave written informed consent. Concurrent biochemotherapy was administered at 21-day intervals. Patients were admitted to an inpatient oncology unit for a 5-day regimen of dacarbazine, vinblastine, cisplatin, IL-2, and IFN-{alpha}. All patients received an identical chemotherapy and biotherapy regimen, but 17 of 90 patients also received tamoxifen. The biochemotherapy regimen consisted of dacarbazine 800 mg/m2 d1-4 i.v.; vinblastine 1.4 mg/m2 d1-4 i.v.; cisplatin 1.4 mg/m2 d1-4 i.v.; IFN-{alpha} 5 mU/m2 d1-5 s.c.; and IL-2 18 mU/m2 d1, 9 mU/m2 d2, and 4.5 mU/m2 d3-4 i.v. Response to treatment was assessed by following standard criteria at that time. Complete response was defined as the disappearance of all clinical evidence of tumor by physical examination and radiographic studies for at least 4 weeks, during which no new lesions appeared. Partial response was defined as a >50% reduction in the sum of products of perpendicular diameters of measurable lesions without the appearance of any new lesions for a minimum of 4 weeks. Stable disease included minor responses and was defined as the following: (a) a <25% increase in the sum of the products of the perpendicular diameters of all measurable lesions or (b) a decrease of 0% to 49% in the sum of the products of the perpendicular diameters of all measurable lesions without appearance of new lesions for at least 8 weeks. Progressive disease was defined as a ≥25% increase in the sum of the products of the perpendicular diameters of any measurable lesions or the appearance of any new lesion. The development of central nervous system metastases was counted as progressive disease regardless of continued response at other disease sites.

Preparation of Peripheral Blood Mononuclear Cell Specimen. Peripheral blood samples were processed to purify peripheral blood mononuclear cells by sedimentation on a Ficoll-Hypaque cushion (Pharmacia, Alameda, CA) and extensive washing in HBSS. Cells were frozen in 40% human AB serum (Gemini Bioproducts, Calabasas, CA), 50% RPMI 1640 (Life Technologies, Grand Island, NY), and 10% DMSO (Sigma, St. Louis, MO) and stored in a liquid nitrogen freezer at –168°C until use.

Genotyping. Genomic DNA was isolated from peripheral blood mononuclear cells using a QiaAmp kit (Qiagen, Valencia, CA). Detection of the T and A alleles at position +874 of IFN-{gamma} gene as well as the A and G alleles at position –1082 in the promoter region of IL-10 gene was carried out using the amplification refractory mutation system-PCR assay as described by Pravica et al. (12) and Perrey et al. (32). Each sample was included in two PCR reactions differing in the allele-specific primer (Table 1), and all PCR reactions included a human growth hormone sequence as an internal control to avoid false-negative results. The primer sequences and the size of PCR products were summarized in Table 1. DNA (200 ng) was amplified in 50 µL mixture consisting of 200 µmol/L each of deoxynucleotide triphosphates (Fisher Scientific, Tustin, CA), 2.5 units HotStar Taq polymerase (Qiagen), 5 µL of 10x PCR buffer (Qiagen), 5 µmol/L allele-specific primers each, and 1.5 µmol/L internal control primers each. The PCR reaction was run as follows: 15 minutes at 95°C (hot start); 10 cycles of 15 seconds at 95°C, 50 seconds at 60°C (for IFN-{gamma} +874A->T polymorphism) and 65°C (for IL-10 –1082G->A polymorphism), and 40 seconds at 72°C; and 20 cycles of 20 seconds at 95°C, 50 seconds at 56°C (for IFN-{gamma} +874A->T polymorphism) and 59°C (for IL-10 –1082G->A polymorphism), and 50 seconds at 72°C. The final PCR product was extended at 72°C for 5 minutes. Polymorphism in PCR amplicons was detected by gel electrophoresis on 2% agarose gel in the presence of ethidium bromide and visualized under UV light.


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  		Table 1 Amplification refractory mutation system-PCR for genotyping IFN-{gamma} (+874A->T) and IL-10 (–1082G->A)

 
Genotypes for XRCC1 (Arg399Gln), XPD (Lys751Gln), and ERCC1 (codon 118) were analyzed using PCR-RFLP techniques. The DNA fragments containing the polymorphisms were amplified and then digested by restriction enzymes as described previously (29, 23, 21) . Briefly, DNA (50 ng) was amplified in a 50 µL mixture consisting of 200 µmol/L each of deoxynucleotide triphosphates, 2.5 units HotStar Taq polymerase, 5 µL of 10x PCR buffer, 5 µmol/L primers each, and water. The PCR was run as follows: after 15 minutes of initial denaturation at 95°C, 30 cycles of 45 seconds at 95°C, 45 seconds at 60°C, 45 oseconds at 72°C, and final extension at 72°C for 5 minutes. The PCR products were digested by endonucleases according to the manufacturer's recommendation (New England Biolabs, Beverly, MA). The digests were separated on 3% agarose gel with ethidium bromide and visualized under UV light. The primer sequences, PCR conditions, and restriction enzymes used for each polymorphism were summarized in Table 2.


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  		Table 2 PCR-RFLP: primers, restriction enzymes, and fragment sizes

 
Genotyping analyses were repeated twice for all patients, and randomly selected PCR-amplified DNA samples for each gene (n = 2 for each genotype) were examined by DNA sequencing to confirm the genotyping results.

Statistical Analysis. Objective tumor response to biochemotherapy and overall survival were the end points considered in this analysis. Patients with complete response and partial response were combined as a "response" group, whereas patients with stable disease and disease progression as a "nonresponse" group. Survival was calculated from the first day of treatment (study entry) until the last follow-up or death from any cause; patients who were alive at the last follow-up were censored at that time.

The association of each polymorphism and each of the following demographic and baseline prognostic factors: age at enrollment of study (≤44, 45-54, or ≥55 years), gender, number of metastatic sites (1-2, 3, or ≥4), performance status (Eastern Cooperative Oncology Group 0 versus 1-2), tamoxifen treatment, and stage (M1a, M1b, or M1c) with survival was analyzed individually using Kaplan-Meier plots and the log-rank test. In the univariate analysis, the relative risk (RR) ratio and its associated 95% confidence interval (95% CI) were based on the log-rank test. Probability of survival at 12 months and its Greenwood SE and median survival and its 95% CI were calculated. Multivariate analysis was done using Cox proportional hazards regression model. Contingency tables and Fisher's exact test were used for the categorical variables to evaluate the association of the polymorphism and baseline data and the response to biochemotherapy. All analyses were done using Statistical Analysis Systems version 8.2 (SAS Institute, Cary, NC) and Epilog Plus software version 1.0 (Epicenter Software, Pasadena, CA).

An internal validation analysis using bootstrapping was done to eliminate the possibility of overfitting or biasing conclusions on a small extreme subset (33). The internal validity of the models was assessed in all steps of data analysis (univariate analysis and multivariate analysis). Five hundred bootstrap samples were generated from the original sample. Each bootstrap sample consisted of 90 observations drawn from the original data set using simple random sampling with replacement. Variables selected in the original analysis retained if associated P < 0.05 in >50% of sample simulation (34, 35).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical Characteristics of Patients and Its Association with Clinical Outcome. A total of 90 stage IV melanoma patients who received a biochemotherapy regimen of CVD plus IL-2 and IFN-{alpha} were included in the present study. The demographic characteristics of these patients are listed in Table 3. Forty-five of 87 (51.7%) of the patients had objective responses to biochemotherapy (20 with a complete response and 25 had a partial response). Adequate information on response was missing for 3 patients. No clinical variables were associated with response to therapy.


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  		Table 3 Association between demographics and overall survival among study patients with stage IV melanoma

 
The median survival for the whole group was 10.2 months. Thirty-seven of 90 (41%) of patients survived >12 months, whereas only 19 of 90 (21%) of patients survived >24 months after initiation of biochemotherapy. In the univariate survival analyses, among all the clinical variables known to have prognostic importance, the number of metastatic sites and lactate dehydrogenase (LDH) were significantly associated with overall survival (log-rank test P = 0.003 and <0.001, respectively; Table 3). Performance status and tumor stage (M1a/M1b versus M1c) was associated with overall survival at the borderline level (log-rank test P = 0.053 and 0.03 respectively), whereas other variables, such as age, sex, or treatment with tamoxifen, were not. These results are consistent with published data (36), which also suggested that the patients included in this study were representative of stage IV melanoma patients. For the multivariate analysis, the number of metastatic sites and LDH were included as control variables.

Association of Single Genetic Polymorphism with Clinical Outcome. Single nucleotide polymorphisms in five genes were analyzed in this study: cytokines IFN-{gamma} (+874T->A) and IL-10 (–1082G->A) and DNA repair genes ERCC1 (codon 118), XRCC1 (Arg399Gln), and XPD (Lys751Gln). No significant association was observed between the presence of any genetic polymorphism and known prognostic variables (e.g., number of metastatic sites, performance status, and LDH) in these patients.

Two end points were considered for clinical outcome—response to therapy and overall survival. An analysis of time-to-progression was also done. The IFN-{gamma} (+874T->A) polymorphism was found to be strongly associated with response to biochemotherapy (P = 0.001). Patients with the A/A or A/T genotype were more likely to respond to biochemotherapy in contrast to those with the T/T genotype (56.3% versus 26.3%). Similarly, the ERCC1 polymorphism at codon 118 was also significantly associated with response to biochemotherapy (P = 0.030). Patients (63.2%) with the T/T or C/T genotype showed a response to biochemotherapy compared with 16.7% with the C/C genotype. IL-10 (–1082G->A), XRCC1 (Arg399Gln), and XPD (Lys751Gln) did not show any association with response to biochemotherapy (Table 4).


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  		Table 4 Association between genomic polymorphisms and overall survival for patients with stage IV melanoma

 
The genotypes were classified as favorable and unfavorable for each polymorphism based on their biological function (level of cytokine production for IFN-{gamma} and IL-10 genes and DNA repair capacity for ERCC1, XRCC1, and XPD) and published reports (12, 18, 27, 29, 30, 37, 38) . When evaluating the association of allele variants with overall survival by univariate analysis, the IFN-{gamma} (+874T->A) T/T genotype (correlating with high level IFN-{gamma} production) was statistically significantly associated with poor overall survival (P < 0.001). The median survival for the T/T phenotype was 7.0 months compared with 11.8 months for the A/A and A/T phenotypes (RR-2.69; Fig. 1). Marginal associations with overall survival were found for ERCC1 and IL-10 gene polymorphisms. The C/C genotype at ERCC1 codon 118 (correlating with a higher level of ERCC1 gene expression) was associated with a shorter survival time with median survival of 7.0 months compared with 10.5 months for the T/T and C/T genotypes (RR-1.80; log-rank test P = 0.049; Fig. 1). Although the association of IL-10 polymorphisms at promoter site –1082 with overall survival did not reach a significant level, IL-10 seemed to be another promising marker. The median survival for the patients with the A/A genotype (correlating with lower level of IL-10 production) was 10.7 months, whereas those for A/G and G/G was 9.8 months (RR-1.54; log-rank test P = 0.065; Fig. 1). XRCC1 and XPD polymorphisms failed to show any significant association with overall survival. Similarly, IFN-{gamma} (+874T->A) polymorphism was significantly associated with progression-free survival (RR-2.23; P = 0.001), whereas ERCC1 codon 118 polymorphism and IL-10 (–1082G->A) polymorphism were not.



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  		Fig. 1 Survival of patients with metastatic melanoma biochemotherapy (dacarbazine, vinblastine, cisplatin, IL-2, and IFN-2) by IFN-{gamma} (+874A->T), IL-10 (–1082G->A), and ERCC1 (codon 118) gene polymorphisms. Vertical hash marks, time of last follow-up for those patients who were still alive at the time of analysis of the data.

 
Association of Multiple Genetic Polymorphisms with Clinical Outcome. Univariate analysis showed that the IFN-{gamma} (+874A->T) gene polymorphism strongly predicted both response and overall survival. However, for patients with single favorable polymorphisms, the median survival was only ~12 months with a 20% of probability of long-term survival at 24 months, which suggested that patients stratified by single polymorphisms were still a heterogenous population in clinical outcome. Therefore, further stratification was required to define subsets of patients who had a more or less favorable outcome.

In the present exploratory analyses, we attempted to combine important gene polymorphisms found in univariate analyses, so that patients could be further stratified into subgroups with more distinct outcomes. Three gene polymorphisms [IFN-{gamma} (+874A->T), IL-10 (–1082G->A), and ERCC1 (codon 118)] were included in the combined analysis. A commonly used scoring approach was used to simplify the combination analysis (39–41). Each gene polymorphism was assigned a score (i.e., 1 for favorable polymorphisms and 0 for unfavorable ones). The scores for each gene polymorphism were added together to make a total score, which ranged from 0 to 3 for each patient.

Patients were stratified into four subgroups with distinct clinical outcomes according to gene polymorphism scores (0, 1, 2, and 3). Patients (68.4%) with a score of 3 responded to biochemotherapy, whereas the response rates for patients with scores 2 and 1 were 58.3% and 23.5%, respectively. No patient with score 0 responded to biochemotherapy (P = 0.007). Patients with total score of 3 showed the longest median survival (24.1 months) followed by those with a score of 2 (11.4 months). Patients (53%) in the group with a score of 3 survived >24 months compared with 16% in the score of 2 group. Patients with total score of 1 or 0 exhibited the worst clinical outcome (median survival 7.0 months; P < 0.001). Only 6% of patients still survived after 24 months in the group with a score of 1, and all patients in the group with score 0 died within 9 months (Table 6; Fig. 2). The associations were significant even after stratification by the clinically significant variables (LDH and the number of metastatic sites; P = 0.029). In other words, patients with more favorable polymorphisms (lower level IFN-{gamma} and IL-10 and lower level of DNA repair) had a higher possibility of overall survival than those with less favorable polymorphisms (higher level of IFN-{gamma} and IL-10 and higher level of DNA repair).


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  		Table 6 Multivariate analysis of IFN-{gamma} (+874A->T), IL-10 (–1082G->A), and ERCC1 (codon 118) polymorphisms and overall survival

 


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  		Fig. 2 Survival of patients with metastatic melanoma biochemotherapy (dacarbazine, vinblastine, cisplatin, IL-2, and IFN-2) by combination ofIFN-{gamma} (+874A->T), IL-10 (–1082G->A), and ERCC1 (codon 118) gene polymorphisms. Vertical hash marks, time of last follow-up for those patients who were still alive at the time of analysis of the data.

 
Multivariate Analysis of Association of Gene Polymorphisms with Clinical Outcome. Multivariate analysis models were constructed to assess the prognostic significance of cytokine gene polymorphisms IFN-{gamma} (+874A->T) and IL-10 (–1082G->A) and DNA repair polymorphism ERCC1 (codon 118) on overall survival. After the addition of the clinical covariates (LDH and the number of metastatic sites), IFN-{gamma} (+874A->T) had independent prognostic significance as a predictor for overall survival (P = 0.003). In contrast, ERCC1 (codon 118) and IL-10 (–1082G->A) polymorphisms failed to show significance (P = 0.20 and 0.24, respectively). The combination of three polymorphisms still conserved the significance power (P = 0.02; Table 5).


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  		Table 5 Association between combined gene polymorphisms of IFN-{gamma} (+874A->T), IL-10 (–1082G->A), and ERCC1 (codon 118) genes and overall survival

 
Model Validation. Selection of a subset of variables for inclusion in a model is always subject to the bias that the final model reflects the patients in the data set better than the target population in general—often because the variables selected and the estimates of the effects are unduly influenced by a small subset of patients with extreme or unique characteristics. An internal validation analysis using bootstrapping was done to evaluate the model selected in terms of the potential for "overfitting" (33). Five hundred bootstrap samples were generated from the original sample. Each bootstrap sample consisted of 90 observations drawn from the original data set using simple random sampling with replacement. The criterion (≥50%) used for inclusions of variables in the models was similar to those in other studies (34, 35).

First, univariate survival analysis (the log-rank test) was conducted for each of five baseline factors and each of five genomic polymorphisms in each of the 500 bootstrap samples. This bootstrap analysis confirmed that IFN-{gamma} (+874A->T), abnormal LDH, and number of metastatic sites were selected by 97%, 96%, and 91%, respectively, of the bootstrap data sets as prognostic variables significantly associated with survival at the 0.05 level. ERCC1 (codon 118) and IL-10 (–1082G->A) were selected by 72% and 57%, respectively, as the genomic polymorphisms significantly associated with survival at the 0.20 level.

Second, multivariate analysis (Cox proportional hazards model) was conducted for each of three genomic polymorphisms categorized as favorable and unfavorable in the 500 bootstrap samples. The bootstrap analysis indicated that the combination of IFN-{gamma} (+874A->T), IL-10 (–1082G->A), and ERCC1 (codon 118) gene polymorphisms adjusted by the number of metastatic sites and abnormal LDH was significantly associated with overall survival at the 0.05 level in 88% of the bootstrap data sets.

In conclusion, the present study showed cytokine gene polymorphisms were associated with clinical outcome for melanoma patients treated with biochemotherapy. The IFN-{gamma} (+874A->T) polymorphism was a strong and independent predictor, whereas the association with the IL-10 (–1082G->A) polymorphism was weaker. The DNA repair ERCC1 (codon 118) polymorphism was an intermediate predictor. The three gene polymorphisms combined were a more powerful predictor of clinical outcome.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present investigation was designed as an exploratory pilot study to assess the value of cytokine gene polymorphisms when used alone or in combination with DNA repair polymorphisms to predict clinical outcome for patients with advanced melanoma treated with biochemotherapy. This was the first study in which the association between genetic polymorphisms and clinical outcome for melanoma patients has been investigated. Defining and refining prognostic markers for advanced melanoma patients is important, because alternative treatments may be chosen without subjecting patients to unnecessary toxic and complex therapy if the analysis predicts a poor outcome with a particular regimen. The analytic strategy was straightforward. First, single nucleotide polymorphisms were examined individually by univariate analysis. Then, multiple gene polymorphisms were combined to obtain more distinct profile because any single nucleotide polymorphism might not adequately account for variability in outcome in melanoma patients. Next, multivariate analysis was done to determine whether gene polymorphism could be an independent predictor for clinical outcome. Lastly, to avoid overfitting due to the relatively small sample size and exploratory nature of this study, an internal validation analysis was done.

Cytokine gene (IFN-{gamma} and IL-10) polymorphisms were chosen for the primary analysis based on their likely impact on treatment with IL-2 and IFN-{alpha}. DNA repair gene (ERCC1, XRCC1, and XPD) polymorphisms were also chosen based on published data indicating their importance for predicting outcome in patients receiving platinum. A functional polymorphism for each gene was selected for analysis based on information in the literature, suggesting an impact on gene expression (27, 29–32). For example, the T allele located at the +874 position from the translation start site of IFN-{gamma} gene is correlated with high IFN-{gamma} production (12), whereas G allele at IL-10 promoter –1082 site was reported to be associated with high IL-10 production following in vitro stimulation of peripheral blood mononuclear cells with concanavalin A (42).

We found that the IFN-{gamma} (+874T->A) polymorphism is a strong and independent predictor for clinical outcome in melanoma patients treated with biochemotherapy. The T/T genotype, which is correlated with high levels of IFN-{gamma}, was associated with poor response to biochemotherapy as well as reduced overall survival (P < 0.001). The IFN-{gamma} (+874A->T) and ERCC1 (codon 118) polymorphisms were significantly associated with response to therapy, whereas none of the clinical variables included in the present study showed an association with response although some clinical variables are strong survival predictors. There may be a correlation between response to therapy and overall survival; however, melanoma factors that are prognostic for survival are not predictive of response to therapy and vice versa. The IFN-{gamma} (+874A->T) polymorphism is strongly associated with overall survival not only in a univariate analysis but also in a multivariate analysis after adjusting for known clinical predictors (LDH, the number of metastatic sites), which suggests that the IFN-{gamma} (+874A->T) polymorphism is an independent indicator superior to standard clinical variables (e.g., LDH, the number of metastatic sites) for overall survival for patients treated with biochemotherapy.

An appreciable level of clinical evidence supports our results. In a phase III randomized trial of IFN-{gamma} as adjuvant therapy in high-risk melanoma patients, there was a trend toward a poorer disease-free and overall survival in patients who received IFN-{gamma} (43). High IFN-{gamma} serum levels were reported to be a negative independent prognostic factor for disease-free survival in melanoma patients (44, 45).

One potential mechanism to explain our findings could be that a low IFN-{gamma} level reflects an effective immune response against the primary tumor. In contrast, high levels could reflect the absence of an effective antimelanoma immune response. IFN-{gamma} has been shown to increase HLA class II expression both on lymphocytes from melanoma patients (9) and on melanoma tumor cells (46–48). Elevated HLA class II antigen expression is found on thicker primary melanoma tumors and melanoma metastases (49–51), and increased HLA class II expression by tumor cells has immunologic effects, including the induction of immune tolerance (52). It is plausible that increased HLA class II expression by melanomas induced by high IFN-{gamma} levels could result in impaired immune recognition of melanoma and increased risk of disease progression. Meanwhile, it has also been reported that IFN-{gamma} can promote tumor-related immune evasion by down-regulating melanoma antigen expression and recognition of melanoma cells by cytotoxic T cells (53). Alternatively, exposure to high IFN-{gamma} levels may have facilitated the development of a population of melanoma cells that were resistant to the immunomodulatory component of the therapy, resulting in reduced antitumor response and clinical progression.

The IL-10 (–1082G->A) polymorphism showed the same trend although it did not reach a statistically significant level (P = 0.065) possibly due to weak association or small sample size. Polymorphisms associated with low IL-10 production were associated with better prognosis. It seems that the single IL-10 gene polymorphism tested herein is a weak predictor for clinical outcome in melanoma patients treated with biochemotherapy, consistent with the role of IL-10 as an immunosuppressive factor. We also found that DNA repair polymorphism ERCC1 was marginally significantly associated with response to biochemotherapy (P = 0.030) as well as with overall survival (P = 0.049). Consistent with our hypothesis, the group with the ERCC1 polymorphism that correlated with low level of DNA repair showed a better outcome (27).

Because biochemotherapy employs cytotoxic agents as well as cytokines that mediate tumor regression by different mechanisms, multiple genes playing roles important for the function of the disparate components of the regimen may account for the overall clinical effect. IFN-{gamma} and ERCC1 gene polymorphisms were significantly associated with response to biochemotherapy, but IL-10 was not. Therefore, we speculated that IFN-{gamma} (strong predictor) and ERCC1 gene polymorphisms (intermediate predictor) might affect initiation of response to biochemotherapy, whereas IL-10 polymorphism (weak predictor) might affect the maintenance of response (durability of response) after completion of biochemotherapy.

When we combined polymorphisms to assess the association of the combination with clinical outcome, IFN-{gamma}, IL-10, and ERCC1 gene polymorphisms together seemed to improve the discrimination of the analysis. Patients with favorable polymorphisms for all three genes (score of 3), who might not only initiate the an antitumor response but also maintain the response, had a significantly higher response rate (68.4%), longer median survival (24.1 months), and higher probability of long-term survival (53% at 24 months) than those in other subgroups. In contrast, all patients with 0 favorable polymorphisms for three genes (score 0), who might neither initiate a response nor maintain it, died within 9 months. Patients with 1 to 2 favorable polymorphisms had an intermediate clinical outcome. These results suggested that multiple genetic polymorphisms combined could generate more accurate prognostic information compared with single gene polymorphisms.

There are several limitations in this study. First, association and correlation may be missed due to the relatively small sample size. Second, the model selected may overfit the data because of the many models considered and tested (internal validity of models). Third, the 90 patients included in this study might not be representative of other stage IV patients receiving similar biochemotherapy (external validity of models). Only confirming with an independent prospective study will address each of these concerns. Fourth, the DNA repair gene polymorphisms were studied in peripheral blood cells because tumor tissue was unavailable for the present study. Studies of repair gene and cytokine expression in tumor tissue will be of interest.

The goal of our study was to make rational patient care decisions and prospectively select patients most likely to respond to therapy. It will be critical to validate our findings in a larger prospective study so we can develop a more comprehensive and rational panel of gene polymorphisms to predict clinical outcome in melanoma patients receiving biochemotherapy. In summary, a single cytokine genetic polymorphism (e.g., IFN-{gamma}) might be useful as a marker for predicting clinical outcome in melanoma patients receiving biochemotherapy. Combinations of polymorphisms from multiple genes seem to provide more important prognostic information, which could accurately identify patients who might benefit and those who would derive no benefit from the use of biochemotherapy.


   ACKNOWLEDGMENTS
 
We thank Dr. Heinz-Joself Lenz and his laboratory personnel for technical assistance and valuable advice.


   FOOTNOTES
 
Grant support: Lucille and Berle Adams Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/ 1/04; revised 10/27/04; accepted 11/ 3/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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