Analysis of N- and K-Ras Mutations in the Distinctive Tumor Progression Phases of Melanoma
2001; Elsevier BV; Volume: 117; Issue: 6 Linguagem: Inglês
10.1046/j.0022-202x.2001.01601.x
ISSN1523-1747
AutoresMarguerite Stas, H. Degreef, Anouk Demunter, Chris De Wolf‐Peeters, Joost J. van den Oord,
Tópico(s)Cancer Genomics and Diagnostics
ResumoMutations 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. 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. cutaneous melanoma denaturing gradient gel electrophoresis degenerated oligonucleotide primed polymerase chain reaction radial growth phase 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., 1984Clark Jr, W.H. Elder D.E. Guerry D.I.V. Epstein M.N. Green M.H. Horn M. A study of tumor progression: the precursor lesions of superficial spreading and nodular melanoma.Hum Pathol. 1984; 15: 1147-1158Abstract Full Text PDF PubMed Scopus (785) Google Scholar 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., 1984Clark Jr, W.H. Elder D.E. Guerry D.I.V. Epstein M.N. Green M.H. Horn M. A study of tumor progression: the precursor lesions of superficial spreading and nodular melanoma.Hum Pathol. 1984; 15: 1147-1158Abstract Full Text PDF PubMed Scopus (785) Google Scholar;Elder, 1999Elder D. Tumor progression, early diagnosis and prognosis of melanoma.Acta Oncol. 1999; 38: 535-547Crossref PubMed Scopus (72) Google Scholar). 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., 1989Clark W.H. Elder D.E. Guerry D. et al.Model predicting survival in stage I melanoma based on tumor progression.J Natl Cancer Inst. 1989; 20: 1893-1904Crossref Scopus (1026) Google Scholar). 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., 1989van'T Veer L.J. Burgering B.M.T. Versteeg R. et al.N-RAS mutations in human cutaneous melanoma from sun-exposed body sites.Mol Cel Biol. 1989; 9: 3114-3116Crossref PubMed Scopus (251) Google Scholar;Koh et al., 1990Koh H.K. Kligler B.E. Lew R.A. Sunlight and cutaneous malignant melanoma: evidence for and against causation.Photochem Photobiol. 1990; 51: 765-779PubMed Google Scholar). UV is a known melanocyte mitogen and induces specific mutations in cellular genes such as p53 and ras (Van der Lubbe et al., 1988Van der Lubbe J.L.M. Rosdorff H.J.M. Bos J.L. Van der Eb A.J. Activation of N-ras induced by ultraviolet irradiation in vitro.Oncogene Res. 1988; 3: 9-20PubMed Google Scholar;Brash et al., 1991Brash D.E. Rudolph J.A. Simon J.A. et al.A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.Proc Natl Acad Sci USA. 1991; 88: 10124-10128Crossref PubMed Scopus (1730) Google Scholar). Activated ras genes have been found in several types of human malignancies, including melanoma (Bos, 1988Bos J.L. The ras gene family and human carcinogenesis.Mutation Res. 1988; 195: 255-271Crossref PubMed Scopus (678) Google Scholar). 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, 1985Marshall C.J. Human oncogenes.in: Weiss R. Teich N. Varmus H. Coffin J. RNA Tumor Viruses. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1985: 487-558Google Scholar). 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, 1984Gilman A.G. G proteins and dual control of adenylate cyclase.Cell. 1984; 36: 577-579Abstract Full Text PDF PubMed Scopus (1107) Google Scholar). 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., 1981Shih C. Padhy L.C. Murray M. Weinberg R.A. Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts.Nature. 1981; 290: 261-263Crossref PubMed Scopus (564) Google Scholar). 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., 1982Dhar R. Ellis R.W. Shih T.Y. et al.Nucleotide sequence of the p21 transforming protein of Harvey murine sarcoma virus.Science. 1982; 217: 934-936Crossref PubMed Scopus (161) Google Scholar;Reddy et al., 1982Reddy E.P. Reynolds R.K. Santos E. Barbacid M. A point mutation is responsible for the acquisition of transforming properties by the T24 human bladder carcinoma oncogene.Nature. 1982; 300: 149-152Crossref PubMed Scopus (867) Google Scholar;Taparowsky et al., 1983Taparowsky E. Shimuzu K. Goldfarb M. Wigler M. Structure and activation of the human N-RAS gene.Cell. 1983; 34: 581-586Abstract Full Text PDF PubMed Scopus (323) Google Scholar;Bos et al., 1985Bos J.L. Toksoz D. Marshall C.J. et al.Amino-acid substitutions at codon 13 of the N-RAS oncogene in human acute myeloid leukemia.Nature. 1985; 315: 726-730Crossref PubMed Scopus (261) Google Scholar;Bar-Eli et al., 1989Bar-Eli M. Ahuja H. Foti A. Cline M.J. N-RAS mutations in T-cell acute lymphocytic leukemia: analysis by direct sequencing detects a novel mutation.Br J Haematol. 1989; 72: 36-39Crossref PubMed Scopus (21) Google Scholar). 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., 2001Demunter A. Ahmadian M.R. Libbrecht L. et al.A novel N-Ras mutation in malignant melanoma is associated with excellent prognosis.Cancer Res. 2001; 61: 4916-4922PubMed Google Scholar). 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., 1989Albino A.P. Nanus D.M. Mentle I.R. Cordon-Cardo C. McNutt N.S. Bressler J. Andreeff M. Analysis of ras oncogenes in malignant melanoma and precursor lesions: correlation of point mutations with differentiation phenotype.Oncogene. 1989; 4: 1363-1374PubMed Google Scholar 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., 1989Albino A.P. Nanus D.M. Mentle I.R. Cordon-Cardo C. McNutt N.S. Bressler J. Andreeff M. Analysis of ras oncogenes in malignant melanoma and precursor lesions: correlation of point mutations with differentiation phenotype.Oncogene. 1989; 4: 1363-1374PubMed Google Scholar). 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., 1994Platz A. Ringborg U. Mansson Brahme E. Lagerlöf B. Melanoma metastases from patients with hereditary cutaneous malignant melanoma contain a high frequency of N-ras activating mutations.Melanoma Res. 1994; 4: 169-177Crossref PubMed Scopus (31) Google Scholar). In a previous study, we analyzed survival data in relation to ras mutations in CM (Demunter et al., 2001Demunter A. Ahmadian M.R. Libbrecht L. et al.A novel N-Ras mutation in malignant melanoma is associated with excellent prognosis.Cancer Res. 2001; 61: 4916-4922PubMed Google Scholar) 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. 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., 1991Schrier P.I. Versteeg R. Peltenburg L.T. Plomp A.C. Veer L.J. van'T Kruse-Wolters K.M. Sensitivity of melanoma cell lines to natural killer cells: a role for oncogene-modulated HLA class I expression?.Semin Cancer Biol. 1991; 2: 73-83PubMed Google Scholar). MOLT-4 cell line was provided by J. Van Pelt (Laboratory of Hepatology, University Hospitals Leuven, Belgium) (Verlaan de Vries et al., 1986Verlaan de Vries M. Bogaard M.E. Van den Elst H. Van Boom J.H. Van der Eb A.J. Bos J.L. A dot-blot screening procedure for mutated RAS oncogenes using synthetic oligonucleotides.Gene. 1986; 50: 313-320Crossref PubMed Scopus (338) Google Scholar). 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. Microdissection was performed on 5 µm 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 20× 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 µl 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). In 44 cases, genomic DNA was isolated from 10 consecutive frozen whole tissue sections of 20 µm thickness by proteinase K digestion and phenol-chloroform extraction according to standard procedures. DOP-PCR was performed on a thermocycler (Perkin Elmer 480) in two separate phases Table I.Table IThe protocol of Dop-PC1RaProtocol modified from this described by Kuukasjärvi et al (1997).ReagentVolumeReactionPreamplification step 10 × high salt bufferb10 × high salt buffer = 260 mM Tris-HCl (pH 9.5); 65 mM MgCl2.1 µlDenaturation for 1 min at 94°C 2 mM dNTP1 µlAnnealing for 1 min at 94°C 10 µM UNI-primer1 µl3 min ramp from 25°C to 74°C ThermoSequenase (32 U per µl) (diluted in dilution buffer delivered with the enzyme 1:2)1 µlExtension for 2 min at 74°C4 cycles in total Digest (Sample 5 µl)5 µl H2O1 µl10 µlSecond amplification step add: 10 × low salt bufferc10 × low salt buffer = 10 mM Tris-HCI (pH 8.3); 50 mM KCI.5 µlDenaturation for 1 min at 94°C 2 mM dNTP4 µlAnnealing for 1 min at 56°C 100 µM UNI-primer0.6 µlExtension for 2 min at 72°C MgCl2 (25 mM)5 µl30 cycles in total H2O24.9 µl Taq-Polymerase LD (5 U per µl)0.5 µl50 µla Protocol modified from this described by Kuukasjärvi et al (1997).b 10 × high salt buffer = 260 mM Tris-HCl (pH 9.5); 65 mM MgCl2.c 10 × low salt buffer = 10 mM Tris-HCI (pH 8.3); 50 mM KCI. Open table in a new tab The four first cycles (preamplification step) were carried out in a 10 µl reaction mixture (using ThermoSequenase, Amersham Pharmacia, Roosendaal, The Netherlands) at low stringency conditions, and were followed by 30 cycles in a 40 µl 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., 1992Telenius H. Nigel P.C. Bebb C.E. Nordenskjold M. Ponder B.A.J. Tunnacliffe A. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.Genomics. 1992; 13: 718-725Crossref PubMed Scopus (1201) Google Scholar). Reagents, volumes, and reaction conditions are shown in Table I, and are slight modifications from the originally published paper ofKuukasjarvi et al., 1997Kuukasjarvi T. Tanner M. Pennanen S. Karhu R. Visakorpi T. Isola J. Optimizing DOP-PCR for universal amplification of small DNA samples in comparative genomic hybridization.Genes Chromosom Cancer. 1997; 18: 94-101Crossref PubMed Scopus (117) Google Scholar. The PCR product was purified (Quiagen Westburg, Leusden, The Netherlands) before further use. 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 µl, containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 μM each dNTP, 0.2 µM 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., 1988Neri A. Knowles D.M. Greco A. McCormick F. Analysis of Ras oncogene mutations in human lymphoid malignancies.Proc Natl Acad Sci USA. 1988; 85: 9268-9272Crossref PubMed Scopus (204) Google Scholar), 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 × 58°C, 2 × 57°C, 2 × 56°C, 2 × 55°C, 2 × 54°C, 2 × 53°C, 2 × 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. 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. Forty microliters of PCR product was dried out and loaded on the gels to run at 170 V for 4 h in 1 × 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. 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).Table IITumor progression in CM: mutation status of the different growth phases of pigment cell lesionsNRHIST.COMM.ACQ.NCNbDifferent growth phases are shown, i.e., radial GP (RGP); invasive RGP (iRGP); vertical GP (VGIP) and associated nevocellular nevi (ncn) [common acquired (comm.acq), congenital (cong.) and dysplastic (dyspi.) ncn] or metastases (META).CONG.NCNbDifferent growth phases are shown, i.e., radial GP (RGP); invasive RGP (iRGP); vertical GP (VGIP) and associated nevocellular nevi (ncn) [common acquired (comm.acq), congenital (cong.) and dysplastic (dyspi.) ncn] or metastases (META).DYSPL.NCNbDifferent growth phases are shown, i.e., radial GP (RGP); invasive RGP (iRGP); vertical GP (VGIP) and associated nevocellular nevi (ncn) [common acquired (comm.acq), congenital (cong.) and dysplastic (dyspi.) ncn] or metastases (META).RGPbDifferent growth phases are shown, i.e., radial GP (RGP); invasive RGP (iRGP); vertical GP (VGIP) and associated nevocellular nevi (ncn) [common acquired (comm.acq), congenital (cong.) and dysplastic (dyspi.) ncn] or metastases (META).IRGPbDifferent growth phases are shown, i.e., radial GP (RGP); invasive RGP (iRGP); vertical GP (VGIP) and associated nevocellular nevi (ncn) [common acquired (comm.acq), congenital (cong.) and dysplastic (dyspi.) ncn] or metastases (META).VGPbDifferent growth phases are shown, i.e., radial GP (RGP); invasive RGP (iRGP); vertical GP (VGIP) and associated nevocellular nevi (ncn) [common acquired (comm.acq), congenital (cong.) and dysplastic (dyspi.) ncn] or metastases (META).METAbDifferent growth phases are shown, i.e., radial GP (RGP); invasive RGP (iRGP); vertical GP (VGIP) and associated nevocellular nevi (ncn) [common acquired (comm.acq), congenital (cong.) and dysplastic (dyspi.) ncn] or metastases (META).Cases with different mutation status in the various growth phases1ALM IVWTcGrowth phase was present and microdissected but revealed a wild-type (WT) sequence for both N-ras exon 1 and 2, 12 Codon 12 mutations were in all cases a transition from GGT (glycine) to GAT (asparagine), 18 Codon 18 mutations were in all cases a transition from GCA (alanine) to ACA (threonine), Three different mutations were observed on our DGGE gel in codon 61, which resulted in substitutions of glutamine by arginine (CGA) (Θ), lysine (AAA) (O) and one that could not be identified by sequencing (?).WTcGrowth phase was present and microdissected but revealed a wild-type (WT) sequence for both N-ras exon 1 and 2, 12 Codon 12 mutations were in all cases a transition from GGT (glycine) to GAT (asparagine), 18 Codon 18 mutations were in all cases a transition from GCA (alanine) to ACA (threonine), Three different mutations were observed on our DGGE gel in codon 61, which resulted in substitutions of glutamine by arginine (CGA) (Θ), lysine (AAA) (O) and one that could not be identified by sequencing (?).Θ2NM IVWTcGrowth phase was present and microdissected but revealed a wild-type (WT) sequence for both N-ras exon 1 and 2, 12 Codon 12 mutations were in all cases a transition from GGT (glycine) to GAT (asparagine), 18 Codon 18 mutations were in all cases a transition from GCA (alanine) to ACA (threonine), Three different mutations were observed on our DGGE gel in codon 61, which resulted in substitutions of glutamine by arginine (CGA) (Θ), lysine (AAA) (O) and one that could not be identified by sequencing (?).1212 O3ALM VWTcGrowth phase was present and microdissected but revealed a wild-type (WT) sequence for both N-ras exon 1 and 2, 12 Codon 12 mutations were in all cases a transition from GGT (glycine) to GAT (asparagine), 18 Codon 18 mutations were in all cases a transition from GCA (alanine) to ACA (threonine), Three different mutations were observed on our DGGE gel in codon 61, which resulted in substitutions of glutamine by arginine (CGA) (Θ), lysine (AAA) (O) and one that could not be identified by sequencing (?).WTcGrowth phase was present and microdissected but revealed a wild-type (WT) sequence for both N-ras exon 1 and 2, 12 Codon 12 mutations were in all cases a transition from GGT (glycine) to GAT (asparagine), 18 Codon 18 mutations were in all cases a transition from GCA (alanine) to ACA (threonine), Three different mutations were observed on our DGGE gel in codon 61, which resulted in substitutions of glutamine by arginine (CGA) (Θ), lysine (AAA) (O) and one that could not be identified by sequencing (?).ΘCases with identical mutation status in the various growth phases4SSM I12125SSM II18186SSM III18 O18 O7SSM III18188SSM III18189SSM III1212121210SSM III181811SSM IV18 Θ18 Θ18 Θ12SSM IV181813SSM IV181814SSM IV18 ?18 ?15SSM IV12121216SSM IVOO17SSM IV12 Θ O12 Θ O12 Θ O18SSM IV12 ?12 ?12 ?19ALM IV12121220ALM IVOOO21ALM IV121222ALM IV12 Θ12 Θ23ALM IV181824NM IVΘΘ25SSM V1212aHistologic type of the CM: acrolentiginous CM (ALM); superficial spreading CM (SSM) and nodular CM (NM).b Different growth phases are shown, i.e., radial GP (RGP); invasive RGP (iRGP); vertical GP (VGIP) and associated nevocellular nevi (ncn) [common acquired (comm.acq), congenital (cong.) and dysplastic (dyspi.) ncn] or metastases (META).c Growth phase was present and microdissected but revealed a wild-type (WT) sequence for both N-ras exon 1 and 2, 12 Codon 12 mutations were in all cases a transition from GGT (glycine) to GAT (asparagine), 18 Codon 18 mutations were in all cases a transition from GCA (alanine) to ACA (threonine), Three different mutations were observed on our DGGE gel in codon 61, which resulted in substitutions of glutamine by arginine (CGA) (Θ), lysine (AAA) (O) and one that could not be identified by sequencing (?). Open table in a new tab aHistologic type of the CM: acrolentiginous CM (ALM); superficial spreading CM (SSM) and nodular CM (NM). 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.Table IIITumor progression in CM: wild-type samplesCases that were microdissectedaDifferent growth phases of the primary CM were dissected, i.e., the radial growth phase (RGP), the invasive radial growth phase (iRGP), the vertical growth phase (VGP), and the associated metastases (META) or nevi (one lentigo benigna, one compound and one dysplastic nevus).HistologyTotal number of casesN°NeviRGPiRGPVGPMETACases where
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