Mutagenesis, Vol. 15, No. 3, 257-260,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Increased frequency of LOH on chromosome 9 in sporadic primary melanomas is associated with increased patient age at diagnosis
Department of Biosciences, Karolinska Institute, Novum, 141 57 Huddinge and 1 Department of Pathology, Huddinge University Hospital, 141 86 Huddinge, Sweden
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Abstract |
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We carried out statistical analysis of the frequency of loss of heterozygosity (LOH) at 10 microsatellite markers on chromosome 9. In 44 microdissected sporadic primary melanomas a comparison of LOH frequency data with other patient data, like age at diagnosis and tumour thickness, showed an interesting correlation between patient age at diagnosis and frequency of LOH on chromosome 9. The patient group with age >72 years at diagnosis (n = 22, mean age 82.3 ± 6.0 years, mean LOH 3.4 ± 2.3) showed significantly increased LOH frequency (OR 3.1, 95% CI 1.8–5.3;
2 test, P < 0.0001) compared with age group 
72 years (n = 22, mean age 56.1 ± 14.5 years, mean LOH 1.8 ± 1.7). A statistically significant increased frequency of LOH (OR 3.5, 95% CI 1.5–7.9; 2 test, P = 0.03 after Bonferroni correction) was found only at marker D9S736 on 9p22 (telomeric to the INK4–ARF locus) relative to other markers on six different chromosomes. No other marker, including those located within the INK4–ARF locus, showed a statistically significant increased frequency of LOH. Our results for the first time show a non-random tendency for increased allelic loss in melanomas with increased patient age at diagnosis, besides supporting the existence of an additional tumour suppressor gene(s) on chromosome 9. |
Introduction |
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Malignant melanoma is a complex disease, involving a large number of genes. The relative importance and involvement of different chromosomal regions found to be altered in melanoma is not clear. Loss of heterozygosity (LOH) studies have shown allelic losses on chromosomes 6 and 10, however, the allelic losses on chromosome 9p appear to be most common in sporadic and familial melanomas (Healy et al., 1996
, 1998). The INK4–ARF locus on 9p21, which harbours the p16 gene, the p15 gene and the p14ARF gene, has been assigned as the major locus conferring melanoma. The p16 gene is deleted or mutated in more than half of cell lines derived from sporadic melanomas (Kamb et al., 1994; Kumar et al., 1998b; Walker et al., 1998). A low frequency of mutations in the p16 gene in conjunction with a high frequency of allelic losses at 9p21 in sporadic melanomas have suggested the presence of other tumour suppressor genes at this locus (Puig et al., 1995; Flores et al., 1996; Ohta et al., 1996; Kumar et al., 1998a, 1999; Ruiz et al., 1998). The presence of an additional tumour suppressor outside the INK4–ARF locus has also been suggested by various studies on different other cancer types (Waber et al., 1997; Wiest et al., 1997).
In the present study we carried out a statistical analysis of the detected frequency of LOH on chromosome 9 at 10 polymorphic microsatellite markers. A comparison of LOH frequency with other data, like patient age at diagnosis, showed an interesting correlation. The overall allelic loss on chromosome 9 in patient age group >72 years showed a significantly increased LOH frequency (OR 3.1, 95% CI 1.8–5.3; 2 test, P < 0.0001) compared with patient age group 72 years. This is the first report showing an association of increased age with allelic losses in any human tumour. Further, we found a statistically increased frequency of LOH only at a telomeric (to p16) marker D9S736 in comparison with total LOH frequency at different markers on six chromosomes.
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Materials and methods |
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Melanoma specimens
Paraffin blocks containing specimens from primary cutaneous melanomas were collected from the archives of the pathology departments of Huddinge University Hospital and Söder Hospital. The regional oncological centre confirmed that the cases were not from individuals registered as members of dysplastic naevus syndrome families.
All the tumours were thick, i.e. exceeding 0.76 mm, and in the vertical growth phase according to Clark's model of tumor progression (Clark et al., 1989). From paraffin blocks of melanomas 10 µm thick sections were cut and placed on glass slides. The sections were cut and carefully dissected under a microscope into tumour and surrounding normal tissues. DNA was extracted by incubation in a proteinase K digestion buffer as described previously (Kumar et al., 1998a).
LOH analysis
LOH analysis was carried out as described previously (Kumar et al., 1999). Briefly, DNA from melanoma and benign tissues were amplified in a 10 µl volume reaction containing 50 mM KCl, 1.5 mM MgCl2, 0.11 mM each dNTP, 0.2–0.3 µM each primer and 0.5 U Taq polymerase. The temperature conditions were either 95°C (denaturation) for 45 s, 55°C (annealing) for 60 s and 72°C (extension) for 45 s or 95°C for 25 s, 55°C for 25 s and 72°C for 45 s. PCR was carried out for 35–40 cycles followed by a final extension at 72°C for 7 min. The primer sequences for the microsatellite markers and their corresponding chromosomal locations were obtained from the Genome Data Base (http://gdbwww.gdb.org). One primer in each set was labelled at the 5'-end with Cy-5 dye. PCR products were electrophoresed on 6% denaturing polyacrylamide gels and detected on an automated sequencer (ALF Express; Pharmacia). The results were analysed using the Fragment Manager software package. Allelic loss was scored in informative cases when a reduction of at least 50% in one allele was observed in the tumour DNA compared with normal DNA.
Statistical analysis
The level of statistical significance of frequency of LOH at each locus was determined by the 2 test. The frequency of LOH at individual markers was compared with total frequency of LOH for all the markers studied on chromosomes 1, 6, 9, 10, 11 and 13. kP values (modified P values) were obtained after Bonferroni correction, i.e. by multiplying P values by the number of comparisons made (43 in the present study). Linear regression was carried out between LOH frequency in the form of fractional allelic loss (FAL, defined as the ratio of number of markers with LOH to total number of informative markers) and patient age at diagnosis. Melanoma were divided into two groups on the basis of patient age at diagnosis (72 years and >72 years; this division gave the same number of cases in each group) and mean LOH (total LOH divided by number of cases in each group) determined. Odds ratio (OR) for the frequency of LOH between the two groups were determined for all the markers individually. Statistical significance for differences in frequencies of LOH between the two age groups was determined by the 2 test (for all markers) and Fisher exact test (for each marker individually). The Pearson correlation and Spearman rank correlation were used in the SAS software package to test the effect of tumour thickness on the observed correlation between the frequency of LOH and the patient age at diagnosis.
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Results |
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The data on frequency of LOH on chromosome 9 showed a correlation (Figures 1 and 2
) with increased patient age at diagnosis (r = 0.37, P = 0.01). The patient group with age at diagnosis 72 years (n = 22, mean age 56.1 ± 14.5 years, mean LOH frequency 1.8 ± 1.7) showed a significantly decreased frequency of LOH (2 test, P < 0.0001; OR 3.1, 95% CI, 1.8–5.3) compared with those diagnosed at >72 years (n = 22, mean 82.3 ± 6.0 years, mean LOH frequency 3.4 ± 2.3) (Figure 1). Of the individual markers, a statistically significant difference between the two age groups was found at only two markers, IFNA (OR [
]; Fisher exact test, P = 0.01) and D9S257 (OR 13.3, 95%CI 1.7–133.8; Fisher exact test, P = 0.002). In the higher age group six melanomas (25, 27, 30, 34, 39 and 41) showed a large deletion, potentially of the entire chromosome, whereas none of the melanomas in the lower age group showed such a large deletion. Correlation analysis of the FAL value on chromosome 9 against age at diagnosis and tumour thickness by Pearson correlation and Spearman rank correlation showed a significant association of increased LOH with increased age (P < 0.05) but not with increased tumour thickness (P > 0.2). The age data showed a weak correlation with tumour thickness (r = 0.29, P = 0.06). No correlation was detected between the frequency of LOH and tumour site (data not shown).
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Statistical analysis of frequency of LOH at individual markers on chromosome 9, when compared with total LOH at all markers on the same chromosome, did not show an increased frequency of LOH after Bonferroni correction of the P values obtained from
2 tests. Chromosome 9 has on average a very high frequency of LOH and, therefore, we used LOH frequency data from other chromosomes as a reference. On comparison of the frequency of LOH at each marker on chromosome 9 with total frequency of LOH at all markers (included in this study) on chromosomes 1, 6, 9, 10, 11 and 13, marker D9S736, located ~300 kb telomeric to the INK4–ARF locus showed a statistically significant increased frequency of LOH (OR 3.5, 95% CI 1.5–7.9; 2 test, P = 0.03 after Bonferroni correction) (Table I). Microsatellite markers D9S942 and D9S974, which are located within the INK4–ARF locus on 9p21, did not show significantly increased frequencies of LOH, although two individual cases of melanoma (3 and 17) did harbour exclusive allelic losses at these loci (Figure 1
). We have also reported mutations in the p16 gene in 10 of these sporadic melanomas (Kumar et al., 1998a, 1999). Three melanomas (5, 24 and 36) showed allelic losses clustered on markers D9S257, D9S280 and D9S287 without loss of 9p markers (Figure 1).
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Discussion |
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The major result of this study is that in sporadic primary melanoma we found a strong association between increased patient age at diagnosis and increased frequency of LOH on chromosome 9. We found that the patient group with age >72 years at diagnosis had a statistically significant higher frequency of LOH than the patient group with age of diagnosis
72 years. Though separately only two markers, IFNA on the p arm and D9S257 on the q arm, showed statistically significant increased frequency of LOH in melanomas with higher patient age at diagnosis. However, six melanoma cases in the patient group with age at diagnosis >72 years showed potential loss of an entire chromosome compared with none in the lower age group. The majority of human cancers are associated with intrinsic chromosomal instability, which has been argued to be non-random and represents loss of some checkpoint (Lengauer et al., 1998). Allelic loss in tumours arises as a result of some somatic event like deletion, mitotic recombination, non-disjunctional chromosomal loss or gene conversion. Events like deletion and mitotic recombination are indicative of chromosomal instability. While allelic losses at specific loci may indicate specific tumour suppressors, more widespread allelic losses possibly manifest general chromosomal instability or aneuploidy, which is distinct from microsatellite instability. Chromosomal aberrations and mutation frequencies in human lymphocytes increase with age (Jones et al., 1995; Ramsey et al., 1995). The frequency of aneuploidic disorders like Down and Klinefelter syndromes also reportedly increase with increased parental age (Connor and Ferguson-Smith, 1997). In general it is known that DNA repair capacity gradually diminishes and the risk of developing sunlight-induced skin cancer increases with age (Grossman, 1997; Wei, 1998). We hypothesize that melanomas with a higher frequency of allelic losses in higher age group patients represent chromosomal instability/aneuploidy due to loss or accumulation of defects in some hypothetical checkpoint with probable origin in the repair machinery.
One of the questions regarding allelic losses on chromosome 9 has been whether these are associated with initiation or progression of melanoma. The reported correlation between tumour thickness and large deletions on chromosome 9 has been cited in support of a role in melanoma progression. However, our results clearly show that larger deletions are correlated with increased patient age at diagnosis rather than tumour thickness. Although previously the frequency of LOH on chromosomes 6q and 10q, and not 9p, has been associated with poor clinical outcomes in melanoma (Healy et al., 1998), the prognostic relevance of increased LOH frequency on chromosome 9 in patients in the higher age group found in this study remains to be determined. However, primary melanomas with larger allelic deletions have been reported to have larger metastatic potential (Puig et al., 1995). It is thus possible that melanomas in the patients with higher age at diagnosis have greater propensity to metastasize.
We found a statistically non-random increased frequency of allelic losses only at marker D9S736 on 9p22, which is telomeric to the well-characterized INK4–ARF locus. The existence of a putative tumour suppressor gene(s) at loci telomeric to the INK4–ARF locus has been suggested by other studies on sporadic melanoma (Ohta et al., 1996). The possibility of homozygous deletion of the INK4–ARF locus along with LOH in the flanking sequences has been put forward as an alternative hypothesis to the presence of an additional tumour suppressor gene(s) on 9p21–22. Studies on chromosome transfer with the INK4–ARF locus microdeleted chromosome 9 (Parris et al., 1999) and haplotype sharing among p16 gene mutation carriers in Dutch families (van der Velden et al., 1999), however, provide further evidence for the existence of an additional tumour suppressor gene in the vicinity of the locus. We did not find a statistically significant frequency of LOH at any of the markers within the INK4–ARF locus. However, two melanomas showed allelic losses confined to the markers within the locus and we have also reported mutations in the p16 gene in a subset of these melanomas (Kumar et al., 1998a, 1999), which support its role in a subset of sporadic melanoma. Clustering of allelic losses within the three markers at 9q22.1–22.3 without any allelic losses at markers on chromosome 9p suggests the probable involvement of a tumour suppressor at this locus in a subset of melanomas. However, none of the markers within the 9q22.1–22.3 locus showed a statistically significant increased frequency of LOH when compared with frequency of LOH at other markers.
In summary, our results show for the first time that in sporadic primary melanoma a statistically significant increased frequency of LOH on chromosome 9 is associated with increased patient age at diagnosis. Our results also support the existence of an additional tumour suppressor locus outside the INK4–ARF locus on chromosome 9p21–22.
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Acknowledgments |
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This study was supported by a grant from the Swedish Cancer Fund, Stockholm, and the EU Environment and Climate Program.
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Notes |
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* To whom correspondence should be addressed. Tel: +46 8 608 9244; Fax: +46 8 608 1501; Email: rajiv.kumar{at}cnt.ki.se
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References |
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Received on October 22, 1999; accepted on January 28, 2000.
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