and their association with clinical course of melanoma

Changes in CDKN2a gene are known to be linked with sporadic melanoma and hereditary predisposition to this cancer. In the Polish population mutations in the coding region of the CDKN2a gene are rather rare, therefore the attention has been focused on polymorphisms and alterations in uncoding regions such as 3' UTR. The aim of this study was to analyze two common polymorphisms, Ala148Thr and 500 C/G, and correlate them with the clinical course of melanoma. DNA from 285 patients was analyzed and found polymorphisms were correlated with the clinical parameters employing statistical methods. The obtained results allow us to conclude: (i) survival times of 500 C/G carriers vs. cumulating proportion surviving was not statistically significant; (ii) CDKN2a polymorphism 500 C/G correlated with Ala148Thr; (iii) no correlation was observed between the 500 C/G polymorphism and age of diagnosis, localization of primary melanoma and survival time; (iv) we did not find correlation between 500 C/G and type of cancer in the family; (v) changes in the CDKN2a gene were not found in patients with second cancer.


INTRoDUCTIoN
The etiology of cutaneous melanoma is heterogeneous and involves genetic predisposition and environmental factors.Melanoma is a genetically complex disease involving a large number of genes.CDKN2a (OMIM*600160) was identified as the first high-penetrance melanoma susceptibility gene linked with family history of melanoma, young age of onset or multiple primary tumors.The CDKN2a-ARF locus is located on chromosome 9p21 and encodes two distinct proteins involved in cell cycle regulation.p16, one of the protein products of the CDKN2a-ARF gene, is a small cyclin-dependent kinase inhibitor.Germline mutations in the CDKN2a appear in vary-ing frequencies from 5 to 50% in different populations and are more frequent in families with more than three cases of melanoma.On the other hand, mutations in the CDKN2a gene are rather rare, and the number of carriers depends on the geographic area, closely connected with such risk factor as level of UV radiation.Such relationship was confirmed by frequently detected mutations in codon 61 of a RAs gene in the CDKN2a variant carriers (Eskandarpour et al., 2003).In the Polish population, mutations in the CDKN2a gene are very rare (Lamperska et al., 2002;Dębniak et al., 2005).Accordingly, more attention has been focused on untranslated regions of the gene (UTRs) and their polymorphisms.Studies carried out on British and Australian populations 2007 K. M. Lamperska and others have demonstrated genetic changes in the untranslated regions, some related to melanoma incidence.Examples include G/T substitution in position -34 in 5' UTR generating a new AUG start codon.In British and Australian families two variants of intron sequences IVS1 +1104 C/A and IVS1 -1104 C/G which both predispose to melanoma were found (Harland et al., 2005).The CDKN2a gene also carries polymorphisms.The most common sites 500 C/G and 540 C/T are located in 3' UTR (Kumar et al., 1998;2001) and 148 Ala/Thr in exon 2. Functional analysis indicated that this amino acid change did not affect p16 ability to inhibit CDK4 enzymatic activity (Ranade et al., 1995;Reymond & Brent, 1995).It is very difficult to directly asses the effect of changes in 3' UTR on p16 mRNA metabolism and its connection with melanoma development and progression.Indirect functional studies such as assessment of correlation of p16 polymorphisms and clinical features of the disease may provide some insight into mechanisms involved.Accordingly, the aim of the present study was to analyze two common polymorphisms in the CDKN2a gene and correlate them with the clinical course of the disease.

MATeRIAls AND MeThoDs
Blood samples were collected from 425 melanoma patients but only in 285 individuals clinical features were assessed.They included: age of first diagnosis, tumor localization, survival time, age of death, and familial history.Clinical parameters were correlated with the diagnosed polymorphisms employing statistical methods.
DNA isolation.DNA was isolated from whole blood samples or from lymphocytes stored in liquid nitrogen using Wizard genomic extraction kit (Promega, WI, USA) according to the manufacturer's instruction.
sequencing analysis.Bands showing atypical mobility shift were cut out from the gel, eluted into water and amplified with the same pair of primers.Products of PCR reaction were purified and served as sequencing template.Sequencing reactions were performed using the fmol sequencing kit (Promega).
statistical analyses.Statistical analysis was performed using univariate logistic regression.We used Shapiro-Wilk and Lilliefors test and then Student' t-test, χ 2 test with Yates' modifications and Fisher's exact test.The Mann-Whitney U-test served as a nonparametric method.The statistical significance of analyzed factors depending on patient survival or death was estimated.Survival probability was estimated by the Kaplan-Meier method.Data showing statistical significance was correlated with survival time.The statistical significance of prognostic factors depending on survival time was assessed by the Cox nonparametric proportional hazard regression model.A P value < 0.05 was considered as statistically significant.The statistical analysis was performed using Statistica for Windows release 6.0.

ResUlTs
In a group of 285 patients with melanoma some had secondary tumors: one patient had multiple primary melanomas (MPM), five had breast cancer, one had colon cancer, one patient had uterus cancer, one had retinoblastoma and finally one had two different cancers: breast and thyroid.Melanoma was localized in 127 cases on the trunk, in 112 on a limb, in 27 cases on the head, in 2 in an eye, in 2 under a nail, one in rectum and in 14 cases the primary localization was unknown.One-hundred and thirty-two patients died during observation, while most of them (104 cases) within 5 years following melanoma diagnosis.The average age of first diagnosis in the examined group was 48.13 years (range: 19 to 80, S.D. 13.58).The group included 155 women (54.39%) and 130 men (45.61%).
Survival of over eight years was observed in seven cases.In eight cases the exact date of death was not known.The correlation between clinical parameters and survival is present in Table 1.The median survival time according to Kaplan-Meier was 90.87 months, lower quartile (25th percentile) was 42.90 months.The curve of survival is showed in Fig. 1.
Fifty-nine patients reported occurrence of cancer in the family.Melanoma was reported by 12 families (20.33%), however, two melanomas in a family were recognized only in two cases.Among other types of cancers there were: uterus cancer in 8 families (13.6%), lung cancer in 17 families (28.8%), stomach in 9 (15.2%),colon in 6 (10%), larynx in 6 (10%), liver in 4 (6.8%),pancreas in 3 (5%), brain in 3 (5%), and single incidence of other types of cancer in 10 families, finally, two patients reported FAM-MM (OMIM#155600).Although 20.7% of patients had cancer incidence in family history, only two cases could be qualified as syndromes predisposing to melanoma: one of familial melanoma malignum and the second FAMMM-PC (OMIM#606719).The correlation between cancer family history and survival time is presented in Table 2.
PCR-SSCP analysis demonstrated the 500 C/G variant in 62 cases and 148 Ala/Thr polymorphism in seven cases.Both changes together were present in five cases, and the correlation between the variants 500 C/G and 148 Ala/Thr was statistically significant (P = 0.0066).Patients with multiple cancers including melanoma did not show polymorphisms in the CD-KN2a gene.All patients with coexisting melanoma and breast cancer were investigated for the presence of mutations in the BRCA1 gene, characteristic for the Polish population (Górski et al., 2000).No changes in BRCA1 were found.DNA from patients reporting breast cancer in the family was also analyzed for mutations in the BRCA1 gene, but such changes were not found.Polymorphism 500 C/G was found in nine patients having cancers in the family, but no correlation with the type of cancer was established.148 Ala/Thr was present in only one patient with a family cancer history, together with the 500 C/G variant.Since 148 Ala/Thr was recognized mostly in DNA from patients with no cancer history in the family, correlation analysis between the variant and the type of cancer in the family was not performed.At the time of analysis, 153 patients were alive while 132 had died.Polymorphism 500 C/G was found in 26 living persons and 37 dead.The correlation between the percentage of dead patients and the 500 C/G polymorphism was found to be statistically significant (P = 0.0252) (Table 3).No correlation was observed for 148 Ala/Thr (P = 0.5608) alone nor for the     The median age of diagnosis of all the patients was 47 years, while for the carriers of the 500 C/G variant it was 50.No correlation between the age of diagnosis and the presence of the 500 C/G variant was found (P = 0.5984).No correlation was found for carriers of the 148 Ala/Thr polymorphism and the age of melanoma diagnosis (P = 0.2358).

DIsCUssIoN
One of the most significant risk factors is the family history of melanoma, reported by about 10% of melanoma patients.In our studies melanoma appeared only in 12 families, but only two reported up to 2 cases in interview.We did not observe a correlation between the occurrence of polymorphisms in CDKN2a and the familial history of melanoma, most likely due to the limited number of patients in the investigated group.For the same reason no correlation was observed for patients with FAMMM and MPM (only one case).Then we correlated recognized polymorphisms with spectrum of cancers in familial history.It is known that the 148 Ala/Thr variant predisposes to melanoma and associates with breast cancer in the Polish population (Dębniak et al., 2005).Moreover, an epidemiologic study has provided evidence of a link between melanoma and breast cancer developing in the same person (Goggins et al., 2004).Carriers of mutations in the BRCA2 gene exhibit increased risk of melanoma while carriers of mutations in CDKN2a have increased risk of breast cancer.We also observed a coincidence between the presence of melanoma and breast cancer in the same patient, but no mutations in CDKN2a and BRCA1 were found.In a population-based study it has been shown that 148 Ala/Thr heterozygous carriers were more likely to have a first-degree relative with a cancer of any type compared to non-carriers: 57% versus 36%, respectively (Dębniak et al., 2005).We did not observe a correlation between 148 Ala/Thr and the occurrence of cancers in the family.The above variant was found only in one patient with a familial history of cancer.We did not find a correlation between the 500 C/G allele and the type of cancer in a patients' family.
Finally, we looked for correlations between the investigated alleles and the clinical course of melanoma.Such analysis was carried out for polymorphic variants found in DNA isolated from melanoma tissue.In tumors showing loss of heterozygosity the polymorphic allele was usually retained.Polymorphisms in 3' UTR are regarded to be connected with a significantly shorter progression time from primary to metastatic disease (P = 0.0071) (Sauroja et al., 2000).We found a correlation between the percentage of patients dying and the 500 C/G variant, however, when the survival time of patients with the C/G variant vs. cumulative proportion surviving were analyzed, the P value was not statistically significant.We did not find a correlation between the presence of the 500 C/ G polymorphism and the age of diagnosis or localization of primary melanoma.
The phenomenon that the 500 C/G variant appearing more frequently in DNA of melanoma patients is still unknown.The association of CDKN2a polymorphisms with melanoma risk is not strong (Aitken et al., 1999) but the frequency of the 500 C/G polymorphism showed to increase with an increasing family risk group.In our investigation we did not answer the question how the 500 C/G variant is correlated with melanoma, but the obtained results allow us to conclude that: (i) survival times of 500 C/G carriers vs. cumulating proportion surviving was not statistically significant; (ii) the 500 C/G polymorphism correlated with 148 Ala/Thr; (iii) no correlation was observed between the presence of the 500 C/G polymorphism and age of diagnosis, localization of primary melanoma and a survival time; (iv) we did not find a correlation between 500 C/G and type of cancer in the family; (v) no changes in a CDKN2a gene were found in patients with a second cancer.

Figure 1 .
Figure 1.survival time of melanoma patients vs. cumulative proportion surviving according to Kaplan-Meier method.

Figure 2 .
Figure 2. survival time of 500 C/G carriers vs. cumulative proportion surviving according to Kaplan-Meier method.Log-Rank Test; P = 0.0531.