Regular Article

Journal of Investigative Dermatology (2001) 117, 1483–1489; doi:10.1046/j.0022-202x.2001.01601.x

Analysis of N- and K-Ras Mutations in the Distinctive Tumor Progression Phases of Melanoma

Anouk Demunter, Marguerite Stas*, Hugo Degreef†, Chris De Wolf-Peeters and Joost J van den Oord

  1. Department of Pathology, Laboratory of Morphology and Molecular Pathology, Leuven, Belgium
  2. *Department of Surgical Oncology, Leuven, Belgium
  3. †Department of Dermatology, University Hospitals, Katholieke Universiteit Leuven, Leuven, Belgium

Correspondence: Dr A. Demunter, Department of Pathology, Laboratory of Morphology and Molecular Pathology, University Hospital St. Rafael, Minderbroederstraat 12, B-3000 Leuven, Belgium. Email: anouk.demunter@uz.kuleuven.ac.be

Received 27 June 2001; Revised 11 September 2001; Accepted 13 September 2001.

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Abstract

Mutations in the ras genes are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in cutaneous melanoma. To investigate whether these mutations occur in early or late tumor progression phases, we searched for point mutations in the N- and K-ras genes in 69 primary cutaneous melanoma, 35 metastases, and seven nevocellular nevi in association with cutaneous melanoma. Lesions were microdissected in order to procure pure tumor samples from the distinctive growth phases of the cutaneous melanoma; the very sensitive denaturing gradient gel electrophoresis technique was used to visualize the mutations, and was followed by sequencing. Point mutations in the N-ras gene but not in the K-ras gene were detected on denaturing gradient gel electrophoresis. Twenty-three primary (33%) and nine metastatic (26%) melanomas showed bandshifts for N-ras. In the majority of cases, mutations occurring in early growth phases (i.e., the "intraepidermal" radial growth phase), were preserved in later growth phases (i.e., the invasive radial growth phase, vertical growth phase, and metastatic phase), which proves the clonal relationship between the successive growth phases. In three cases, however, the mutations differed between the distinctive growth phases within the same cutaneous melanoma, due to the occurrence of an additional mutation (especially in codon 61) in a later tumor progression phase. Our approach also permitted us to analyze the mutational status of nevi, associated with cutaneous melanoma. Six out of seven associated nevi carried the same sequence (mutated or wild-type) as the primary cutaneous melanoma, whereas in one case the sequence for N-ras differed between the primary melanoma and the associated nevus. In conclusion, this approach allowed us to demonstrate the clonal relationship between subsequent growth phases of melanoma and associated nevi; our results suggest that N-ras exon 1 mutations preferentially occur during early stages of tumor progression and hence may be involved in melanoma initiation, whereas those in N-ras exon 2 are found preferentially during later stages and hence are more probably involved in metastatic spread of cutaneous melanoma.

Keywords:

carcinogenesis, DGGE, melanoma, microdissection, Ras

Abbreviations:

CM, cutaneous melanoma; DGGE, denaturing gradient gel electrophoresis; DOP-PCR, degenerated oligonucleotide primed polymerase chain reaction; RGP, radial growth phase; VGP, vertical growth phase

Tumor progression is the process by which gradual accumulations of irreversible genetic alterations result in increasing malignancy, which is reflected by invasion, metastasis, and resistance to therapy. This process occurs in discrete, sequential steps.Clark et al (1984) suggested that in cutaneous melanoma (CM) the successive phases of tumor progression are both clinically and histologically recognizable. The epidermal radial growth phase (RGP) is the phase in which the cancer spreads in the epidermis resulting clinically in an irregular enlargement and color variegation of the macular pigment cell lesion. The microinvasive RGP is characterized by invasion of the dermis by single melanoma cells or small nests of nonproliferating tumor cells. The vertical growth phase (VGP) is histologically characterized by the appearance of expansile nodules in the dermis that may clinically be accompanied by the appearance of a nodule. The metastatic phase is characterized by dissemination of tumor cells to lymph nodes or distant organs (Clark et al, 1984;Elder, 1999). Clinical follow-up of large cohorts of CM patients has shown that the malignant melanocytes in the first two phases (i.e., the intraepidermal and invasive RGP) do not have the competence for metastasis (Clark et al, 1989).

The key processes underlying tumor progression are activation of oncogenes, inactivation of tumor-suppressor genes, and impaired DNA reparation. Ultraviolet radiation (UV) has been causally linked to melanoma initiation and progression (van't Veer et al, 1989;Koh et al, 1990). UV is a known melanocyte mitogen and induces specific mutations in cellular genes such as p53 and ras (Van der Lubbe et al, 1988;Brash et al, 1991). Activated ras genes have been found in several types of human malignancies, including melanoma (Bos, 1988). The three members of the ras gene family are the N-ras gene, located at chromosome 1, the H-ras gene, located at chromosome 11, and the K-ras gene at chromosome 12 (Marshall, 1985). Ras proteins share biochemical properties with G-proteins, known to play a role in the signal transduction pathway from membrane-bound receptors to adenylate cyclase (Gilman, 1984). All activating mutations in ras genes have in common that they convert the normal ras proteins into proteins that are capable of transforming NIH/3T3 cells (Shih et al, 1981). Mutations in naturally occurring ras oncogenes have been found in codons 11, 12, and 13 of exon 1 and in codons 59 and 61 of exon 2 (Dhar et al, 1982;Reddy et al, 1982;Taparowsky et al, 1983;Bos et al, 1985;Bar-Eli et al, 1989). Apart from these well-characterized mutations, we recently described a novel mutation in codon 18 of N-ras exon 1 in a subset of CM with excellent prognosis (Demunter et al, 2001).

In CM, mutations in ras oncogenes have repeatedly been found although their role in the pathogenesis of CM still remains poorly understood. An extensive investigation byAlbino et al (1989) revealed mutations in 24% of cultured CM, in contrast to 5%-6% in noncultured primary and metastatic melanoma. Except for one mutation in codon 13, all of these were located in codon 61 of N-ras, which appears to be the favorable region of mutations, due to the preferential formation of cyclobutane dimers at this site following UV (Albino et al, 1989). Other studies have shown a similar N-ras mutation frequency in cultured melanoma cells and a much lower frequency in primary CM or metastases; K- and H-ras mutations occur rarely in CM (Platz et al, 1994).

In a previous study, we analyzed survival data in relation to ras mutations in CM (Demunter et al, 2001) using whole tissue extracts. Unfortunately, approaches that use extracts from whole tumors, or cell lines derived from an unknown growth phase, do not allow study of the chronology of genetic alterations associated with these discrete stages of tumor progression in CM. Therefore, in this study, we started from the same material as in our previous study but used microdissection to obtain DNA samples from histologically identified tumor progression phases. This technique allowed us not only to analyze the chronology of Ras mutations during the progression of CM, but also to demonstrate the clonal relationship between subsequent growth phases.

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Materials and methods

Materials

Cell lines
 

Two cell lines [NCI-H23 (CRL-5800) and HCT-116 (CCL-247)] with mutations in K-ras exon 1 were obtained from ATCC. N-ras transfectants 11A15 and 7D8 and melanoma cell line Mel-634 were kindly provided by C. Aarnoudse (Department of Clinical Oncology, University Hospital, Leiden, The Netherlands) (Schrier et al, 1991). MOLT-4 cell line was provided by J. Van Pelt (Laboratory of Hepatology, University Hospitals Leuven, Belgium) (Verlaan de Vries et al, 1986).

Patient material
 

This study is based on 69 primary CM and 35 metastases. From 22 patients, both the primary CM and the metastasis were available for analysis. Seven CM were associated with a contiguous nevus, including one congenital nevus, one lentigo simplex, two compound nevocellular nevi, and three sporadic dysplastic nevocellular nevi. In order to overcome problems related to fixation artifacts, both frozen and paraffin-embedded tissue sections from the same lesions were used. At first, DNA extractions from whole tissue sections were used to screen the cases for point mutations. When the denaturing gradient gel electrophoresis (DGGE) gel, capable of detecting a single point mutation, indicated the presence of a mutation in the form of an abnormal migration pattern, the distinctive growth phases of the CM were further microdissected to evaluate the mutation status of the different tumor progression phases. In addition, microdissection was performed on several cases (nevi, primary CM, and metastases) with a wild-type banding pattern on DGGE in order to confirm the sensitivity of the technique, and to exclude the occurrence of false-positive results due to the polymerase chain reactions (PCRs). As controls, cell lines harboring well-defined Ras mutations were used.

Methods

Microdissection
 

Microdissection was performed on 5 microm thick buffered formalin-fixed, paraffin-embedded tissue sections as well as on frozen sections in cases showing well recognizable, distinctive tumor progression phases Figure 1. After staining with hematoxylin and eosin and confirmation of the presence of two or more growth phases, both frozen and paraffin-embedded consecutive tissue sections were digested by incubation at 40°C for 3 h in collagenase H (Boehringer Mannheim, Brussels, Belgium). Different phases of the pigment cell lesions (nevus, "intraepidermal" RGP, microinvasive RGP, VGP, and metastatic phase) were carefully microdissected with a sterile needle under an inverted microscope (Leica DM IL) using a 10 or 20times objective. In 12 CM, more than one clone per growth phase was selected. The slides were placed in a Petriplate and covered with sterile water. Dissection was joystick-controlled. A total of 20–100 cells were collected from a single growth phase using serial tissue sections in each case. The cells were aspirated with another sterile glass needle, transferred to an Eppendorf tube and resuspended in 5 microl of a solution (260 mM Tris-HCl pH 9.5; 65 mM MgCl2) containing 7 mg per ml proteinase K (Boehringer Mannheim, Brussels, Belgium). Samples were incubated overnight at 55°C followed by boiling for 1 min to inactivate proteinase K. All material was used for degenerated oligonucleotide primed PCR (DOP-PCR).

Figure 1.
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Microdissection of the distinctive growth phases of MM. Frozen section of a SSMM Clark level IV showing the various growth phases, i.e. ''pure'' RGP (1), the ''invasive'' RGP (2) and the VGP (3) before (A) and after (B) microdissection

Full figure and legend (263K)

DNA extractions
 

In 44 cases, genomic DNA was isolated from 10 consecutive frozen whole tissue sections of 20 microm thickness by proteinase K digestion and phenol-chloroform extraction according to standard procedures.

DOP-PCR
 

DOP-PCR was performed on a thermocycler (Perkin Elmer 480) in two separate phases Table I.


The four first cycles (preamplification step) were carried out in a 10 microl reaction mixture (using ThermoSequenase, Amersham Pharmacia, Roosendaal, The Netherlands) at low stringency conditions, and were followed by 30 cycles in a 40 microl reaction volume (using AmpliTaq polymerase LD, Perkin-Elmer Applied Biosystems, Lennik, Belgium) at high stringency conditions. Both PCRs contained the UN1-primer (5'-CCGACTCGAGNNNNNNATGTGG-3', with N = A, C, G, or T) allowing universal amplification of genomic DNA (Telenius et al, 1992). Reagents, volumes, and reaction conditions are shown in Table I, and are slight modifications from the originally published paper ofKuukasjarvi et al (1997). The PCR product was purified (Quiagen Westburg, Leusden, The Netherlands) before further use.

Specific PCR
 

The regions centering on codons 12, 13, and 61 of the K- and N-ras genes were selectively amplified using PCR. Thermal cycling was carried out with the GeneAmp PCR system 9600 (Perkin Elmer) in final volumes of 50 microl, containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 muM each dNTP, 0.2 microM of each primer (for N-ras exon 1, 5'-forward-CTGGTG TGAAATGACTGAGT-3', 5'-reverse-[GC]-GGTGGGATCATATTC ATCTA-3'; for N-ras exon 2, 5'-forward-GTTATAGATGGTGAAACCTG, 5'-reverse-[GC]-ATAC ACAGAGGAAGCCTTCG; for K-ras exon 1, 5'-forward-CCT GCTGAAAATGACTGAAT-3', 5'-reverse-[GC]-TGTTGGATCAT ATTCGTCCA-3'; for K-ras exon 2, 5'-forward-GTAATTGAT GGAGAAACCTG-3'; 5'-reverse-[GC]-ATACACAAAGAAAGCCC TCC-3') (Neri et al, 1988), 500 ng of DNA, and 2.5 U of Taq polymerase (AmpliTaq Gold, Perkin Elmer). A 40 bp GC-clamp was attached to the reverse primer ([GC] = GCCCGCCGCGC CCCGCGCCCGGCCCGCCGCCCCCGCCCG) and proved to be sufficient for the demonstration on DGGE of the cell line mutations in both exons of K- and N-ras. The amplification protocol (K-ras and N-ras exon 1) consisted of 40 cycles with denaturation at 94°C, annealing at 50°C, and extension at 72°C for 1 min. An initial denaturation step of 94°C for 10 min and a final incubation at 72°C for 2 min were included. A touchdown PCR was used for K- and N-ras exon 2, to eliminate nonspecific product. After the initial denaturation at 94°C for 10 min, a two-cycle protocol was used with denaturation for 30 s followed by an annealing at 58°C for 30 s. The next two cycles had an annealing at 57°C and so on. After 14 cycles (2 times 58°C, 2 times 57°C, 2 times 56°C, 2 times 55°C, 2 times 54°C, 2 times 53°C, 2 times 52°C) another 30 cycles were added with a denaturation at 94°C for 30 s, annealing at 50°C for 45 s, and an extension at 72°C for 60 s. A final incubation at 72°C for 2 min was also included.

DGGE

To test the DGGE conditions determined by this approach, control mutations were analyzed (NCI-H23, HCT-116 from the ATCC and some melanoma cell lines kindly provided by C. Aarnoudse, Department of Clinical Oncology, Leiden, The Netherlands). A 12% polyacrylamide gel containing a 20%-50% gradient of urea and formamide was sufficient to detect all of the cell line mutations. To demonstrate the sensitivity of the DGGE assay, the mutant PCR product was mixed with the corresponding wild-type PCR product, heated, and allowed to re-anneal to generate heteroduplexes, i.e., hybrids formed between mutant and wild-type DNA strands Figure 2a. DNA from MOLT-4 cell line (heterozygous for mutation in N-ras codon 12 position 1) was serially diluted with normal human spleen and revealed a detection sensitivity of 2.5% mutant DNA in the wild-type sample Figure 2b.

Figure 2.
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Sensitivity of the DGGE mutation detection assay. (A) To demonstrate the sensitivity of the DGGE, a mutant sample (lane 2) (codon 18 mutation) was mixed with a wild type sample (lane 3) to generate heteroduplexes (lane 1). Mutations were clearly resolved by showing a band pattern deviant from that of the wild type. (B) DNA from MOLT-4 cell line (heterozygous for mutation in N-RAS codon 12 position 1) was serially diluted with normal human spleen and revealed a detection sensitivity of 2.5% mutant DNA in wild type DNA. Lane 1 to 9 represents the dilution of the MOLT-4 cell line (100%, 80%, 60%, 40%, 20%, 10%, 5%, 2.5%, and 0% in lane 9). Lane 10 shows a wild type pattern of a wild type sample.

Full figure and legend (31K)

Forty microliters of PCR product was dried out and loaded on the gels to run at 170 V for 4 h in 1 times TAE buffer kept at a constant temperature of 60°C. After electrophoresis, the gel was stained with ethidium bromide and photographed by UV transillumination. The bands were excised from the gel and purified with QIAEX II (Quiagen, Westburg). DNA sequencing was done to confirm and identify the point mutations.

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Results

DNA from 111 cases (69 CM, 35 metastases, and seven nevi) was subjected to selective amplification of the K- and N-ras sequences using the PCR/DGGE approach. Screening of DNA extracted from whole tissue sections revealed wild-type sequences on DGGE in 74 out of these 111 cases (46 CM, 26 metastases, and two nevi). In the remaining 37 cases, point mutations in the N-ras gene but not in the K-ras gene were detected on DGGE (Table II, Figure 3).

Figure 3.
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N-ras gene point mutations observed on a DGGE gel. A 20-50% DGGE gel showing the migration pattern for 9 samples in N-ras exon 2 (lanes 1 to 9) and 2 samples with aberrations in N-ras exon 1 (lane 10 and 11). Case 1 to 4 showing a transversion from CAA (glutamine) to CTA (leucine) in codon 61 of N-ras exon 2. Lanes 5 and 8 exhibit a change from CAA (glutamine) to AAA (lysine) in codon 61 of N-ras exon 2. Lanes 6, 7 and 9 contain wild type samples. Lanes 10 and 11 show mutations in codon 12 of N-ras exon 1 (GGT (glycine) to GAT (asparagine) transition).

Full figure and legend (177K)


To confirm the findings on the whole tissue extract, mutation analysis in microdissected growth phases of 28 wild-type cases (21 primary CM and seven metastases) was performed Table III.


Twenty-three primary CM (33%) and nine (26%) metastatic melanomas showed bandshifts for N-ras. Of these cases, six CM and two metastases showed mutations in both N-ras exon 1 and 2. Fifteen CM and three metastatic melanoma showed only N-ras exon 1 mutations, whereas N-ras exon 2 bandshifts were found in two primary CM and four metastatic melanoma Table II. To confirm our results and to exclude false negative results due to fixation and paraffin-embedding, or due to too small amounts of DNA after microdissection, the DNA of six CM was analyzed both after extraction from whole tissue sections and from microdissected cells, and both approaches were applied to frozen and formalin-fixed, paraffin-embedded tissue. Identical point mutations were obtained using these different techniques. In the 12 cases in which more than one clone from the RGP or VGP was selected, identical results were obtained for the different clones.

The N-ras exon 1 mutations consisted of codon 12 mutations in which GGT (glycine) was substituted for GAT (asparagine) and codon 18 mutations in which GCA (alanine) was mutated to ACA (threonine) (Demunter et al, 2001). The cases with this codon 18 mutation are the same cases as previously reported in a separate paper revealing the functional significance of this mutation (Demunter et al, 2001). No codon 13 mutations were seen. The normal amino acid at codon 61 of the N-ras gene is glutamine (CAA). Three different mutations in this codon were observed on our DGGE gel, which resulted in substitutions of glutamine by arginine (CGA), lysine (AAA), and one that could not be identified by sequencing.

Following microdissection of the distinctive growth phases, 22 primary CM showed identical point mutations in N-ras in subsequent phases of tumor progression, i.e., in an associated nevus and the "intraepidermal" RGP (cases 4, 9, 11, and 18; Table II), in the "pure" and "microinvasive" RGP (cases 5 and 12; Table II), in the RGP and VGP (17 cases; Table II), and in the VGP and metastasis (cases 9, 15, 17, 19, 20, and 24; Table II).

Moreover, in seven cases, more than two subsequent growth phases could be microdissected (cases 11 and 18 with a nevus, RGP, and VGP; cases 15, 17, 19, and 20 with an RGP, VGP, and metastasis; and case 9 with a nevus, RGP, VGP, and metastasis), and in all these cases identical point mutations were found throughout tumor progression. In three primary CM, however, the DGGE results varied between the various tumor progression stages, due to the emergence of mutations in later growth phases. Cases 1 and 3 had wild-type sequences in the RGP and VGP of their primary CM, but revealed a codon 61 mutation in their metastasis. One nodular Clark IV CM (case 2; Table II) showed a codon 12 mutation in the VGP, and an additional codon 61 mutation emerged in its metastasis.

Anatomical site and mutation status were compared in the 69 primary CM. In the group of primary CM with wild-type sequences, 46% of the CM were from sun-exposed areas; 28% were situated at covered body areas; and 12% of unknown origin. The group primary CM with mutations showed similar data; 48% were from sun-exposed areas; 30% from covered body sites; and in 5% the site of origin was not known.

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Discussion

In this study, we used microdissection of paraffin-embedded and frozen sections in combination with a very sensitive PCR technique and DGGE gels to detect point mutations in the K- and N-ras exon 1 and 2 regions in 69 primary CM, 35 metastases, and seven nevi. This technique proved to be very reliable, as described by others (Bernsen et al, 1998), as identical results were obtained with DNA extracted from frozen as well as from paraffin-embedded tissue and with DNA obtained from whole tissue samples as well as from single cells, microdissected from the same sample.

Using this same detection system, we recently discovered a new mutation in codon 18 of N-ras exon 1 in primary CM associated with an excellent prognosis (Demunter et al, 2001). Apart from this novel mutation, 32 out of 104 cases (primary CM and metastases; 31%) revealed one or more mutations in the N-ras gene with DGGE and sequencing. This is in close agreement with the Ras mutation rate of 36% detected byBall et al (1994) using dot blot hybridization on 100 primary and metastatic melanoma. No K- or H-ras mutations were detected in our series. Our findings confirm that ras mutations preferentially affect the N-ras (Albino et al, 1989;Platz et al, 1994).

Of the 32 mutated melanomas in our series, eight cases had mutations in both N-ras exon 1 (codons 12 and 18) and N-ras exon 2 (codon 61) and all other cases showed mutations in either exon 1 or 2 of the N-ras gene. In accordance with previous data no codon 13 mutations were found in our study (Van der Lubbe et al, 1988). All codon 12 mutations in our samples consisted of a transition from glycine to asparagine. The majority of the codon 61 mutations were substitutions from the normal amino acid into either a lysine or arginine, substitutions known to convert the normal p21 protein into one with transforming potential (Barbacid, 1987;Bos, 1988). In contrast to previous studies, no correlation was found between the mutation status of the CM and the anatomical site (i.e., sun-exposed or covered areas of the body) (Van der Lubbe et al, 1988;Jiveskog et al, 1998).

It has been questioned whether mutations in ras genes are an early or a late event in the development of CM. A few reports describe ras mutations in primary CM associated with sun exposure (Van Elsas et al, 1995;Jiveskog et al, 1998), suggesting that ras mutations are involved in CM initiation. Other studies have concluded that most of the activating ras mutations occur during progression in CM, and that ras mutations are not required for CM initiation (Albino et al, 1989). Our study is the first to locate ras mutations within distinctive tumor progression phases. In our series, 23 of the microdissected primary CM displayed an RGP, and 21 of the neoplastic cells from this phase revealed N-ras mutations Table III. The presence of N-ras mutations in the earliest stage of tumor progression in CM, i.e., the pure RGP, lends support to the hypothesis that mutations in ras contribute to the initiation of CM. Although the RGP does precede the VGP in CM, one can never exclude the possibility that successive clones may outgrow the original clones within a CM and that in some cases cells from the VGP may repopulate the RGP. It may therefore be difficult to be certain that N-ras mutations arose at a particular stage of the CM. Different clones per growth phase, however, were picked in 12 CM of our series and revealed identical results for each growth phase.

Following microdissection of 25 CM, the majority showed identical point mutations between the different growth phases of the primary CM, as well as between the primary CM and the subsequent metastasis Table III. In cases 5 and 12 Table II, DNA could be isolated from both the pure and the invasive RGP. The latter stage of tumor progression is characterized by invasion of the dermis by single CM cells or by small clusters of nonproliferating cells and should be differentiated from the expansile VGP, as tumor cells in the invasive RGP are considered not to have metastasizing potential despite their apparent invasiveness; patients with CM in this stage indeed carry an excellent prognosis (Clark et al, 1989). The tumor cells in the invasive RGP are considered to display the same biologic characteristics (Kath et al, 1989) and the same phenotype as those in the "intraepidermal" RGP (Guerry et al, 1993). Our results show identical mutations [alanine (GCA) to threonine (ACA)] in codon 18 of N-ras exon 1 in both the pure and invasive RGP, and thus suggest that, in addition to the phenotype, also the genotype is shared by these two early growth phases.

In six cases (case 9, 15, 17, 19, 20, and 24), the mutations found in the metastasis were identical to those occurring in the VGP. Previous studies have shown that the VGP and metastatic phase share many features, including chromosomal abnormalities and phenotype (Albelda et al, 1990). Our data prove the clonal derivation of the metastasis from the VGP and thus confirm previous cell biologic data that CM cells in the VGP have metastatic potential.

In five cases (case 9, 15, 17, 19, and 20) we were able to isolate neoplastic cells from the RGP, VGP, and the metastasis, and in these five cases an identical set of N-ras mutations was found throughout the successive phases of tumor progression. These data prove the clonal relationship between the successive growth phases and confirm the data ofWiltshire et al (1995) who used microdissection and comparative genomic hybridization to illustrate the clonal relationship between successive growth phases in three CM. Our results also confirm the data ofNakayama et al (2001) who demonstrated genetic clonality between primary CM and subsequent in-transit metastases in 25 patients, based upon the analysis of loss of heterozygosity.

In three CM, an additional mutation, not noted in the primary CM, emerged in a later tumor progression phase. Two primary CM (cases 1 and 3, Table II) with wild-type ras genes showed a codon 61 mutation in their metastasis; one nodular CM (case 2, Table II), with a codon 12 mutation in the VGP and metastasis, presented an additional codon 61 mutation in the metastasis. Although it cannot be completely ruled out that these codon 61 mutations were already present in a small population of tumor cells in an earlier tumor progression phase, the sensitivity of our technique renders this unlikely, and suggests that during tumor progression new mutations have been acquired by neoplastic cells. These findings are in agreement with recent data fromShellman et al (2000) who suggested an important role for activated ras in the progression of CM, and lend support to the model ofClark et al (1984) in which CM is supposed to progress in a stepwise manner along distinctive growth phases. This stepwise tumor progression not only involves activation of oncogenes, but also inactivation or loss of tumor-suppressor genes. In a recent loss of heterozygosity study comparing primary CM and matched in-transit metastases using eight microsatellite DNA markers on six chromosomes, half of the patients were found to demonstrate an additional loss of heterozygosity in an in-transit metastasis (Nakayama et al, 2001).

Taken together, our data show a high incidence of N-ras exon 1 mutations in primary CM; in particular, codon 18 mutations were found to be related with the early growth phases of CM. In contrast, mutations in codon 61 were more frequently observed in metastases than in primary CM. These findings may suggest a role for N-ras exon 2 mutations in later stages of melanoma progression and a role for N-ras exon 1 mutations in early CM development.

Our approach also permitted us to analyze the mutational status of nevi associated with CM. The exact place of common and dysplastic nevi within the scheme of tumor progression of CM is still debated, and few studies have searched for ras mutations in nevi. Albino et al found no mutations in common acquired and dysplastic nevi (Albino et al, 1989). These cases were not associated with primary CM, however, and do not permit investigation of the clonal relationship between the two lesions.Carr and Mackie (1994) investigated congenital nevi and dysplastic nevi: whereas no mutations were found in dysplastic nevi, a high frequency of N-ras codon 61 mutations was found in the congenital nevi. In our study, six out of seven nevi carried the same sequence (mutated or wild-type) as the contiguous primary CM. Only in case 2, a nodular CM with a codon 12 mutation in its VGP, did the associated nevus show a wild-type genotype. Three nevi, associated with superficial spreading melanoma (SSM), showed the histologic and cytologic features of dysplastic nevi. Two of the dysplastic nevi (case 4 and 11, Table II) showed the same N-ras mutation as the adjacent CM, whereas the third dysplastic nevus Table III and the adjacent CM were free of mutations. One congenital nevus was included in the study (case 18, Table II) and revealed the same set of mutations as the contiguous primary CM. Although the number of cases studied is still small, the finding of N-ras mutations in CM and adjacent congenital or dysplastic nevi in some of our cases is in line with a previous study (Lee et al, 1997) suggesting that dysplastic nevi and CM share their cellular response to some mutagenic agents (e.g., UV light). In addition, the finding of identical mutated sequences in nevi and contiguous CM suggests a clonal relationship between the two types of lesions and the possibility that the nevus served as precursor of the CM.

In conclusion, we have demonstrated that N-ras mutations, particularly in exon 1, may occur in the earliest phases of tumor progression of CM and in nevi associated with CM. Mutations, if present, are mostly preserved throughout tumor progression, which proves the clonal relationship between the different growth phases of CM. Additional mutations, especially those in codon 61, may occur during tumor progression and may play one of the roles in the development of metastasis in CM.

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Acknowledgments

This study was accomplished with the financial support of grant OT/98/33 of the Katholieke Universiteit Leuven to A.D. The authors thank Miet Vanherck and Monique Pattou for assistance in the Molecular Pathology Laboratory.

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