Multiple primary melanoma revisited

BACKGROUND: Incidence of cutaneous melanoma continues to increase in the Caucasian population worldwide. Approximately 5% of melanoma patients develop additional primary melanoma. This rate is significantly higher than the estimated lifetime risk of an individual for developing the disease (1.4%). These features suggest that a genetic predisposition may underlie multiple primary melanomas (MPMs). Prior studies had identified CDKN2A mutations in a few MPM individuals. The objectives of this study were to determine the frequency of family history of melanoma in MPM cases, to characterize other clinical features including history of other cancer, and to determine the association with functional CDKN2A mutations. METHODS: This study used a case series design. All living patients who had been seen in the Pigmented Lesion Clinic at the University of Pennsylvania and who had more than one primary invasive malignant melanoma or an invasive primary followed by an in situ melanoma were eligible for participation. RESULTS: Individuals with MPM frequently had a family history of melanoma, dysplastic nevi (DN), and/or another cancer including basal cell carcinoma (BCC), and squamous cell carcinoma breast cancer, and a personal history of DN, and basal cell carcinoma. Germline mutations in CDKN2A gene were identified in 8 of 96 MPM cases (8.3%, 95% confidence interval, 6.7-9.9%). CONCLUSIONS: These data indicate that the presence of MPM is associated with a modest incidence of a family history of melanoma, DN, or BCC and a small association with CDKN2A mutations. Therefore, in addition to the MPM index case, other family members can benefit from screening and regular surveillance for melanoma, DN, and BCC.     

High frequency of multiple melanomas and breast and pancreas carcinomas in CDKN2A mutation-positive melanoma families

BACKGROUND:: Inherited mutations in the CDKN2A tumor suppressor gene, which encodes the p16(INK4a) protein, and in the cyclin-dependent kinase 4 (CDK4) gene confer susceptibility to cutaneous malignant melanoma. We analyzed families with two or more cases of melanoma for germline mutations in CDKN2A and CDK4 to elucidate the contribution of these gene defects to familial malignant melanoma and to the occurrence of other cancer types. METHODS:: The entire CDKN2A coding region and exon 2 of the CDK4 gene of an affected member of each of 52 families from southern Sweden with at least two cases of melanoma in first- or second-degree relatives were screened for mutations by use of polymerase chain reaction-single-strand conformation polymorphism analysis. Statistical tests were two-sided. RESULTS:: CDKN2A mutations were found in 10 (19%) of the 52 families. Nine families carried an identical alteration consisting of the insertion of arginine at position 113 of p16(INK4a), and one carried a missense mutation, in which the valine at position 115 was replaced with a glycine. The 113insArg mutant p16(INK4a) was unable to bind cdk4 and cdk6 in an in vitro binding assay. Six of the 113insArg families had at least one member with multiple primary melanomas; the 113insArg families also had a high frequency of other malignancies-in particular, breast cancer (a total of eight cases compared with the expected 2.1; P =.0014) and pancreatic cancer (a total of six cases compared with the expected 0.16; P<.0001). Families with breast cancer also had a propensity for multiple melanomas in females, suggesting that a sex-dependent factor may modify the phenotypic expression of CDKN2A alterations. CONCLUSIONS:: Our findings confirm that the majority of CDKN2A-associated melanoma families in Sweden are due to a single founder mutation. They also show that families with the CDKN2A 113insArg mutation have an increased risk not only of multiple melanomas and pancreatic carcinoma but also of breast cancer.     

CDKN2A germ-line mutations in individuals with multiple cutaneous melanomas

Germ-line CDKN2A mutations are present in some kindreds with hereditary cutaneous melanoma, and in Sweden a founder mutation with an extra arginine in codon 113 (113insR) has been identified. We screened 80 individuals with at least two primary cutaneous melanomas, who were identified mainly by a search of a regional cancer registry, for germ-line CDKN2A mutations. In nine patients, CDKN2A alterations that may contribute to melanoma predisposition were detected. In six individuals with a family history of melanoma, the 113insR founder mutation was present. One patient, who also had a family history of melanoma, had a 24-bp deletion that included codons 62-69. An in vitro binding assay established that the resulting mutant p16 protein was unable to bind cyclin-dependent kinase 4 and cyclin-dependent kinase 6. Two patients without a family history of melanoma had CDKN2A alterations: (a) one had a mutation in the 5' noncoding sequence (-14C/T); and (b) the other had an insertion of an extra T in codon 28, which results in a stop signal in codon 43. The median age at diagnosis of the first melanoma was significantly lower, the number of primary melanomas was significantly higher, and the presence of a family history of melanoma was significantly more common in patients with CDKN2A mutations than in those without germ-line mutations. The proportion of CDKN2A mutation carriers was significantly higher among patients treated for three or more primary melanomas compared with those with two tumors only. We conclude that mutation screening of individuals with multiple primary melanomas is a useful strategy to identify new melanoma kindreds with CDKN2A germ-line mutations.     

Association of MC1R variants and risk of melanoma in melanoma-prone families with CDKN2A mutations.

Major risk factors for melanoma include many nevi, especially dysplastic nevi, fair pigmentation, freckling, poor tanning ability, and germ line mutations in the CDKN2A, CDK4, or MC1R genes. We evaluated the relationship between MC1R and melanoma risk in CDKN2A melanoma-prone families with extensive clinical and epidemiologic data. We studied 395 subjects from 16 American CDKN2A families. Major melanoma risk factors were assessed by clinical examination or questionnaire; MC1R was sequenced. Odds ratios were estimated by unconditional and conditional logistic regression models. We examined the distribution of MC1R variants and median ages at melanoma diagnosis in multiple primary melanoma (MPM) and single primary melanoma (SPM) patients. Presence of multiple MC1R variants was significantly associated with melanoma, even after adjustment for major melanoma risk factors. All 40 MPM patients had at least one MC1R variant; 65% of MPM patients versus only 17% of SPM patients had at least two MC1R variants (P < 0.0001). For all 69 melanoma patients combined, as well as the 40 MPM patients, there was a statistically significant decrease in median age at diagnosis as numbers of MC1R variants increased (P = 0.010 and P = 0.008, respectively). In contrast, no significant reduction in age at melanoma diagnosis was observed for SPM patients (P = 0.91). The current study suggests that the presence of multiple MC1R variants is associated with the development of multiple melanoma tumors in patients with CDKN2A mutations. Additional studies are needed to confirm these findings and to explore the mechanisms that may contribute to this relationship.     

CDKN2A and CDK4 variants in Latvian melanoma patients: analysis of a clinic-based population

Germline mutations of the CDKN2A and CDK4 genes explain a significant proportion of familial melanoma. To date, there have been few published estimations of the prevalence of such mutations in sporadic melanoma patients. In this study, we investigated CDKN2A and CDK4 exon 2 for germline mutations in 125 consecutive cutaneous malignant melanoma patients recruited through the Latvian Oncological Center, using amplicon melting analysis and sequencing. No disease-related CDKN2A germline mutations were identified in any of the melanoma patients analysed but the previously described CDK4 mutation, Arg24His, was found in one patient with a family history of melanoma. CDKN2A polymorphisms were studied as putative low penetrance susceptibility genes. The proportion of cases with polymorphisms in this Latvian melanoma population was Ala148Thr (c.442G>A) (6%), 500 C/G (c.*29C>G) (18%), and 540 C/T (c.*69C>T) (20%); however, only the frequency of the Ala148Thr polymorphism was higher in melanoma patients than in 203 controls (6 versus 1%, P=0.03). Ala148Thr has also been reported in association with melanoma in a Polish series but not in an English series. We therefore examined the Ala148Thr carrier's haplotype in 10 Latvian and 39 Polish samples. No significant difference was seen between these populations and the predominant haplotype observed in English samples, giving no indication that the discrepancy could be explained by population differences in linkage disequilibrium. In summary, our results show that germline mutations at the CDKN2A locus are rare in sporadic melanoma in Latvia. The study does, however, provide some additional evidence for a role for the CDKN2A polymorphism Ala148Thr as a low penetrance susceptibility gene. The detected CDK4 exon 2 mutation was found in only the seventh family identified worldwide with a germline CDK4 mutation.     

The contribution of large genomic deletions at the CDKN2A locus to the burden of familial melanoma

Mutations in two genes encoding cell cycle regulatory proteins have been shown to cause familial cutaneous malignant melanoma (CMM). About 20% of melanoma-prone families bear a point mutation in the CDKN2A locus at 9p21, which encodes two unrelated proteins, p16(INK4a) and p14(ARF). Rare mutations in CDK4 have also been linked to the disease. Although the CDKN2A gene has been shown to be the major melanoma predisposing gene, there remains a significant proportion of melanoma kindreds linked to 9p21 in which germline mutations of CDKN2A have not been identified through direct exon sequencing. The purpose of this study was to assess the contribution of large rearrangements in CDKN2A to the disease in melanoma-prone families using multiplex ligation-dependent probe amplification. We examined 214 patients from independent pedigrees with at least two CMM cases. All had been tested for CDKN2A and CDK4 point mutation, and 47 were found positive. Among the remaining 167 negative patients, one carried a novel genomic deletion of CDKN2A exon 2. Overall, genomic deletions represented 2.1% of total mutations in this series (1 of 48), confirming that they explain a very small proportion of CMM susceptibility. In addition, we excluded a new gene on 9p21, KLHL9, as being a major CMM gene.     

The prevalence of CDKN2A germ-line mutations and relative risk for cutaneous malignant melanoma: an international population-based study.

Germ-line mutations of CDKN2A have been identified as strong risk factors for melanoma in studies of multiple-case families. However, an assessment of their relative risk for melanoma in the general population has been difficult because they occur infrequently. We addressed this issue using a novel population-based case-control study design in which "cases" have incident second- or higher-order melanomas [multiple primary melanoma (MPM)] and "controls" have incident first primary melanoma [single primary melanoma (SPM)]. Participants were ascertained from nine geographic regions in Australia, Canada, Italy, and United States. In the 1,189 MPM cases and 2,424 SPM controls who were eligible and available for analysis, the relative risk of a subsequent melanoma among patients with functional mutations who have an existing diagnosis of melanoma, after adjustments for age, sex, center, and known phenotypic risk factors, is estimated to be 4.3 (95% confidence interval, 2.3-7.7). The odds ratio varied significantly depending on the type of mutation involved. The results suggest that the relative risk of mutation carriers in the population may be lower than currently believed and that different mutations on the CDKN2A gene may confer substantially different risks of melanoma.     

Melanoma candidate genes CDKN2A/p16/INK4A, p14ARF, and CDK4 sequencing in patients with uveal melanoma with relative high-risk for hereditary cancer predisposition

The reported frequencies of germline mutations in the melanoma candidate genes are low in patients with uveal melanoma (UM). However, the number of families studied is limited and the majority of the published reports used low-sensitivity techniques for mutational screening. Identifying the frequency of alterations in any of the melanoma genes in patients with UM with increased hereditary cancer risk is important for proper counseling of these patients. We studied a total of 47 patients with UM including three with a family history of UM, 18 with a family and/or personal history of cutaneous melanoma (CM), three with early age at diagnosis (<30), 11 with increased risk for a known familial cancer syndrome, and 12 with a second primary tumor. Germline screening for mutations in CDKN2A, p14ARF, and exon 2 of CDK4 was carried out by direct sequencing. We identified a variant (IVS1-69 C>T) of uncertain significance in exon 1b of p14ARF in one of the patients with UM and his mother who also had UM. The variant was neither detected in any of the other patients with UM nor in 146 controls. We did not identify pathogenic mutations in CDKN2A nor exon 2 of CDK4 gene. Our study supports the low frequency of germline mutation of the CM candidate genes in patients with UM with family histories suggestive of a high risk for hereditary cancer. Germline testing for CDKN2A might be reserved for patients with UM with a family history of two or more CM.     

Factors predicting the occurrence of germline mutations in candidate genes among patients with cutaneous malignant melanoma from South Italy

Clinical predictors for germline mutations of candidate genes in large clinic based population of patients with cutaneous malignant melanoma (CMM) are widely awaited. Using denaturing high-performance liquid chromatography (DHPLC) analysis and DNA sequencing, 557 consecutively-collected CMM patients originating from South Italy were screened for CDKN2A germline mutations; subsets of them were screened for mutations in the BRAF and BRCA2 genes. Seven CDKN2A mutations were detected in 14 (2.5%) CMM patients. Relative risk of carrying a CDKN2A mutation for CMM patients was demonstrated to significantly increase with the presence of familial recurrence of melanoma (risk ratio (RR)=6.31; p=0.0009), multiple primary melanomas (RR=3.43; p=0.0014), and early onset age (RR=4.56; p=0.0026). All CDKN2A mutations were observed in non-Sardinian patients (14/441; 3.2%), whereas BRAF and BRCA2 genes were found mutated in Sardinian patients (3/116; 2.6%). Such indicators of the presence of CDKN2A mutations will be useful in counselling patients about undergoing genetic testing. Our findings strongly suggest that mutation rates of candidate cancer genes may deeply vary among CMM patients from different geographical areas.     

A single nucleotide polymorphism in the 3'untranslated region of the CDKN2A gene is common in sporadic primary melanomas but mutations in the CDKN2B, CDKN2C, CDK4 and p53 genes are rare.

In this report we present the results of mutational analysis of the CDKN2B, CDKN2C, CDK4, p53 genes and 5'UTR of the CDKN2A gene in a set of 44 sporadic primary melanomas, which had been earlier analysed for mutations in the CDKN2A (p16/p14(ARF)) gene. No tumour-associated mutations were detected except in 1 melanoma where we found a CC>T* deletion-mutation in the codon 151-152 (exon 5) of the p53 gene. On the basis of our preliminary results, we did extended genotyping of the 500 C>G and 540 C>T polymorphisms in the 3'UTR of the CDKN2A gene in 229 melanoma cases and 235 controls. The T-allele frequency (for 540 C>T polymorphism) in melanomas was significantly higher than in controls (0.14 vs. 0.08; chi(2) = 5.95, p = 0.01; OR = 1.71, 95%CI = 1.11-2.66). The heterozygote frequency for this polymorphism was 0.26 (59/229) in melanomas compared to 0.13 (30/235) in healthy controls (chi(2) = 11.4; p = 0.0007; OR = 2.34, 95% CI = 1.40-3.92). The frequency of the 500 C>G polymorphism in the 3'UTR in the CDKN2A gene was not significantly higher in melanomas compared to healthy controls. The 500 C>G polymorphism, however, was in linkage disequilibrium with approximately 50 kb apart the C>A intronic polymorphism in the CDKN2B gene (determined in 44 melanomas and 90 controls; Fisher exact test, p<0.0001). Finally, the sequence analysis of genomic DNA isolated from T cell lymphocytes of healthy individuals exhibited that the codon reported as last of exon 2 of the CDKN2C gene is rather the first codon of exon 3.     

The CDKN2A tumour suppressor gene: no mutations detected in patients with melanoma and additional unrelated cancers

Germ-line mutations of the CDKN2A tumour suppressor gene have been reported in association with familial melanoma, sporadic melanoma with multiple primary lesions and also pancreatic cancer. We studied the hypothesis that patients with melanoma and additional unrelated cancers may harbour mutations in the CDKN2A gene. Twenty seven patients with histologically confirmed melanoma who also had additional cancers such as breast, colorectal, lymphoma and other neoplasms were studied. We also examined 17 additional patients, 13 of whom had a first-degree relative with melanoma and four who had two or more primary melanomas. Some patients belonged to more than one of these categories. No mutations of the CDKN2A tumour suppressor gene were detected among patients with melanoma and additional cancers. The previously described Met53Ile CDKN2A mutation located in exon 2 was detected in a female patient with melanoma metastatic to the regional lymph nodes, multiple primary cutaneous lesions, atypical naevi and a first-degree relative with melanoma. The studied cohort is too small for firm conclusions. However, it would appear that melanoma and additional, apparently unrelated, cancers developing in the same individual are likely to be related to a combination of low-risk susceptibility genes and environmental factors.     

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.     

A natural history of melanomas and dysplastic nevi: an atlas of lesions in melanoma-prone families

BACKGROUND: Few long-term clinical and histologic data for melanocytic lesions have been available based on the mutation status of families at an increased risk of melanoma. In the current study, the authors describe the clinical and histologic features of dysplastic nevi and melanoma over time in families at an increased risk of melanoma with differing germline mutations in CDKN2A, CDK4, or not yet identified genes. METHODS: Thirty-three families with > 2 living members with invasive melanoma were evaluated clinically and followed prospectively for up to 25 years. All the participants were evaluated by the same study team at the Clinical Center of the National Institutes of Health or in local clinics. After informed consent was obtained, family members (n = 844) were examined and photographed. Blood was obtained for genetic studies; genotyping for CDKN2A and CDK4 was performed. Sequential photographs of melanocytic lesions were taken as part of the clinical evaluations. When melanocytic lesions were removed, the histology was reviewed. Representative photographs and photomicrographs were selected for six classes of lesions and three mutation groups. RESULTS: All the families were found to have members with dysplastic nevi and melanoma; 17 had mutations in CDKN2A, 2 had mutations in CDK4, and 14 had no mutations in either gene identified. The majority of dysplastic nevi either remain stable or regress; few change in a manner that should cause concern for melanoma. With careful surveillance, melanomas can be found early. CONCLUSIONS: The melanomas and dysplastic nevi that were found to occur in the study families did not appear to vary by the type of mutation identified in the families.     

p16/CDKN2 and CDK4 gene mutations in sporadic melanoma development and progression

The p16/CDKN2 (MTSI) gene encoding for the p16 inhibitor of cyclin D/CDK4 complexes is frequently mutated and deleted in a large fraction of melanoma cell lines, and p16 germline mutations have also been observed in familial melanomas. Moreover, a CDK4 gene mutation, responsible for a functional resistance of CDK4 kinase to p16 inhibitory activity, has been described to occur in some cases of familial melanoma. These data strongly support the idea that deregulation of the CDK4/cyclin D pathway, via CDKN2 or CDK4 mutations, is of biological significance in the development of melanoma. To shed light on the role of these alterations in the development and progression of sporadic melanoma, 12 primary melanomas and 9 corresponding metastases were analyzed for CDKN2 and CDK4 gene mutations. Of the 12 primary melanomas analyzed, 4 showed the presence of mutational inactivation of the p16 protein and 2 carried silent mutations. No metastases showed the presence of CDKN2 mutations, indicating that mutations of this cyclin-dependent kinase inhibitor is not common in the progression of sporadic melanoma. On the other hand, the absence, in the metastases, of the CDKN2 mutation detected in the corresponding primary tumors suggests that 9p21 homozygous deletion may play a major role in the metastatic spreading of this type of tumor. None of the cases analyzed showed the presence of an Arg24Cys mutation, which functionally protects CDK4 from p16 inhibition. This indicates that CDK4 mutation plays a minor role in the development and progression of sporadic melanoma. Int. J. Cancer 74: 26–30. © 1997 Wiley-Liss, Inc.     

Early onset may predict G101W CDKN2A founder mutation carrier status in Ligurian melanoma patients

Although the presence of multiple cases of melanoma on the same side of a family is the best predictor of germline CDKN2A mutation, other features (i.e. early age at onset) may be useful to identify carriers. We analysed the records of 682 hospital-based Ligurian melanoma patients. Of these, 238 cases (34 familial, 14 non-familial multiple primary and 190 non-familial single primary melanomas) were consecutively enrolled for screening of the CDKN2A and CDK4 genes. Screening of the 34 familial patients revealed that nine were carriers of the CDKN2A G101W founder mutation. Of the 14 non-familial multiple primary melanoma patients, three carried the G101W founder mutation and one the P48T mutation. For the non-familial patients with a single melanoma, 17 of 190 carried germline CDKN2A mutations, with most (16/17) carrying the G101W Ligurian founder mutation and one a novel single base pair substitution, D74Y. The effect of mutation on age at diagnosis was significant (P=0.012) after correcting for melanoma type (familial or non-familial), number of primaries (single or multiple), gender and disease occurrence (incident or prevalent). Early age at onset may be a good predictor of CDKN2A mutation in Liguria, where the G101W founder mutation is prevalent among melanoma patients, independent of family history.     

BRCA1, BRCA2, TP53, and CDKN2A germline mutations in patients with breast cancer and cutaneous melanoma

PURPOSE: From epidemiological studies it appears that breast cancer (BC) and cutaneous melanoma (CMM) in the same individual occur at a higher frequency than expected by chance. Genetic factors common to both cancers can be suspected. Our goal was to estimate the involvement of "high risk" genes in patients presenting these two neoplasia, selected irrespectively from family history and age at diagnosis. EXPERIMENTAL DESIGN: Eighty two patients with BC and CMM were screened for BRCA1, BRCA2, TP53, CDKN2A and CDK4 (exon 2) germline mutations. RESULTS: Deleterious mutations were identified in 6 patients: two carriers of a BRCA1 germline mutation, two carriers of TP53 germline mutations (one of which also harbored a BRCA2 deleterious mutation, the other one a BRCA2 unclassified variant), and two carriers of a CDKN2A germline mutation. In addition, 6 variants of unknown signification were identified in BRCA1 or BRCA2 genes. Regarding family history, 3/13 (23%) patients with a positive family history of BC or CMM were carriers of a germline mutation, whereas only 3/69 (4%) patients without family history were carriers of a germline mutation. CONCLUSION: Our findings show that few patients with BC and CMM who lacked family histories of these cancers are carriers of deleterious germline mutations in four of the five genes we examined. We describe for the first time, two simultaneous BRCA2 and TP53 mutations, suggesting that analysis in more than one gene could be performed if a patient's personal or familial history does not match a single syndrome.     

Mutations in the TP53 gene in human malignant melanomas derived from sun-exposed skin and unexposed mucosal membranes

Mutations in the p53 tumour suppressor gene ( ) have been linked to several types of cancer. We therefore investigated whether such mutations occur in malignant melanomas and, if so, whether they are linked to ultraviolet (sun) light exposure. For the first time, mutations in mucosal membranes and adjacent tissues shielded from sunlight were compared with those in cutaneous melanomas from sun-exposed skin. Archival tissues were obtained from 35 patients with a primary melanoma taken from unexposed mucosal areas and from 34 patients with a primary melanoma located in chronically sun-exposed head and neck skin. was characterized by means of polymerase chain reaction amplification and single-strand conformation polymorphism assay followed by nucleotide sequencing. The results showed that 17.6% of the primary cutaneous and 28.6% of the primary mucosal melanomas had point mutations in. Among the cutaneous melanomas, one showed three mutations in exon 7, and one had two mutations in exon 5; the mutation was in the same allele in both cases. One mucosal melanoma had two mutations in exon 7, both in the same allele, and another had two mutations, one in exon 7 and one in intron 6, both in the same allele. C<--T mutations at dipyrimidine sites, considered fingerprints for ultraviolet light-induced mutations, were about equally distributed among patients with melanomas from chronically sun-exposed areas (six out of nine; 67%) and those with melanomas from unexposed mucosal areas and adjacent skin (eight out of 14; 57%). Our data, demonstrating the presence of such mutations even in melanomas from mucosal membranes, clearly suggest that factors other than, or additional to, ultraviolet radiation are operational in the induction of mutations in melanomas.     

Mutational analysis of the CDKN2 gene in metastases from patients with cutaneous malignant melanoma.

We analysed 26 metastases from 25 patients with sporadic cutaneous malignant melanoma for alterations in the CDKN2 gene by a combined polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP)/nucleotide sequencing approach. Eleven alterations (one in exon 1, five in exon 2 and five in the 3'' non-coding sequence of the exon 3 region) were concordantly and independently detected by both SSCP and nucleotide sequence analysis. Two of the exon 2 changes and the five changes in the non-coding exon 3 region are likely to represent natural polymorphism. Four (15%) of 26 metastases thus had CDKN2 mutations and belonged to 3 (12%) of 25 patients. Semi-quantitative PCR furthermore revealed no sign of homozygous deletions of the CDKN2 exon 2 region. The results support an involvement of the CDKN2 product in the development of a subgroup of sporadic melanomas and encourage the search for alterations in additional genes of the 9p21 region.     

BRAF alterations are associated with complex mutational profiles in malignant melanoma

To evaluate the mutational profiles associated with BRAF mutations in human melanoma, we have studied BRAF, RAS, PTEN, TP53, CDKN2A and CDK4 genes and their expression in melanoma lesions. Owing to the lack of sufficient material from fresh specimens, we employed short-term cell lines obtained from melanoma biopsies. In all, 41 melanoma obtained from eight primary lesions, 20 nodal, 11 cutaneous and two visceral metastases from patients with sporadic (n=31), familial (n=4) and multiple melanoma (n=2) were analysed. The results revealed novel missense mutations in the BRAF, PTEN, CDKN2A and CDK4 genes. Overall, activating mutations of BRAF and loss of functional p16 and ARF were detected in the majority of melanomas (29/41, 36/41 and 29/41, respectively), while PTEN alterations/loss, NRAS and TP53 mutations occurred less frequently (6/41, 6/41 and 10/41, respectively). In the resulting 12 mutational profiles, p16/ARF loss associated with mutated BRAFV599E was the most represented (n=15). In addition, TP53 and PTEN mutations were always accompanied with BRAF alterations, while PTEN loss was found in association with CDKN2A or TP53 mutations in the absence of BRAF activation. The p16/ARFDelta+BRAF/RAS profile was significantly associated with a longer survival, while complex mutational profiles were detected in highly aggressive disease and poor survival. These data support the existence of several molecularly defined melanoma groups which likely reflect different clinical/biological behaviour, thus suggesting that a more extensive molecular classification of melanoma would significantly impact its clinical management.