The frequency patterns of CTLA-4 alleles were first evaluated in the healthy control and melanoma populations. There were no statistical differences in the incidence of CTLA-4 polymorphisms between melanoma patients and healthy controls (Table ), except for JO 31, where the T/T allele was higher in controls (33.3% vs 24.3%) while G/G and G/T was lower (p = 0.047).
Frequencies of CTLA-4 polymorphisms in melanoma patients and healthy controls
Patient demographics and baseline characteristics have been described elsewhere [15
]. With a median follow up of 70.7 months [only among patients alive (censored values), range 7.1-138.7 months], there were 158 recurrences (median RFS 55 months, range 1 to 115 months) and 105 deaths (median OS not reached yet, range 2 to 86 months).
RFS and OS did not differ significantly between patients with the alleles represented by these polymorphisms. Τhe corresponding p-values for RFS and OS are presented in Table and Figures ,,,,,. In addition, RFS and OS did not differ significantly in the cohort of patients with AG 49 GG when compared with patients with AG49 AA or AG (p = 0.5 and p = 0.51 respectively). No differences were again demonstrated when CT 318 CC and CT 60 GG where3 compared with the cohort of patients either heterozygous or homozygous to the protective allele (p = 0.38 and p = 0.58, and p = 0.92 and p = 0.38 respectively).
Univariate Cox Regression Models of Relapse-free Survival and Overall Survival
High association between the different polymorphisms was found (Fisher's exact p-value < 0.001 for all associations). Genotypes corresponding to the six CTLA-4 polymorphisms did not significantly deviate from the Hardy-Weinberg equilibrium. The test indicates significant linkage disequilibrium among the six polymorphisms
We analyzed the segregation pattern of CT 318, AG 49, CT 60, JO 27, JO 30, JO 31 SNPs on 572 chromosomes and identified 5 major haplotypes (table ). No statistically significant differences for RFS or OS were found for the presence of each of the 3 most common haplotypes.
CTLA-4 most frequent haplotypes
The association of Cw*06 with the CTLA-4 alleles was investigated and a statistically significant association was found with AG 49 (p = 0.023). In patients with positive Cw*06, 61.8% were AG 49 AA, 29.1% were AG 49 AG and 9.1% were AG 49 GG. The median relapse-free survival for Cw*06 positive patients with genotype AG 49 AG was 76.4 months and has not been reached yet for genotypes AA and GG. In Cw*06 negative patients the median relapse-free survival for genotypes AG49 AA, AG and GG was 56.7, 36.2 and 24.6 respectively. Median overall survival has not been reached yet in Cw*06 positive patients in all three genotypes (AG 49 AA, AG, GG) and in the Cw*06 negative cohort it was 86. 1, 66.7 and 61.2 months, respectively. However, no statistically significant differences were found for HLA Cw*06 positive patients in terms of RFS or OS, among AG 49 groups (p = 0.62 and p = 0.46 respectively). Likewise, no statistically significant differences were found for HLA Cw*06 negative patients in terms of RFS and OS among A/G 49 groups (p = 0.42 and p = 0.39 respectively). RFS and OS did not differ significantly in the cohort of patients with AG 49 GG vs AG/AA positive for HLA Cw*06 (p = 0.37 and p = 0.23 respectively) or negative for HLA Cw*06 (p = 0.22 and p = 0.22 respectively). In the cohort of patients included in the prospective autoimmune study, CTLA-4 polymorphisms were investigated in 157 out of 200 patients (48 autoimmunity group and 109 without evidence of autoimmunity). No statistically significant association was found among any of the six polymorphisms investigated. In the multivariate Cox model for RFS and OS, HLA Cw*06 and autoimmunity were statistically significantly correlated with RFS (p = 0.043 and p < 0.001 respectively), while only autoimmunity was found to be statistically significant for OS (p = 0.001).