John A. Curtin, Ph.D., Jane Fridlyand, Ph.D., Toshiro Kageshita, M.D., Hetal N. Patel, M.S., Klaus J. Busam, M.D., Heinz Kutzner, M.D., Kwang-Hyun Cho, M.D., Setsuya Aiba, M.D., Ph.D., Eva-Bettina Bröcker, M.D., Philip E. LeBoit, M.D., Dan Pinkel, Ph.D., and Boris C. Bastian, M.D.
Background Exposure to ultraviolet light is a major causativefactor in melanoma, although the relationship between risk andexposure is complex. We hypothesized that the clinical heterogeneityis explained by genetically distinct types of melanoma withdifferent susceptibility to ultraviolet light.
Methods We compared genome-wide alterations in the number ofcopies of DNA and mutational status of BRAF and N-RAS in 126melanomas from four groups in which the degree of exposure toultraviolet light differs: 30 melanomas from skin with chronicsun-induced damage and 40 melanomas from skin without such damage;36 melanomas from palms, soles, and subungual (acral) sites;and 20 mucosal melanomas.
Results We found significant differences in the frequenciesof regional changes in the number of copies of DNA and mutationfrequencies in BRAF among the four groups of melanomas. Samplescould be correctly classified into the four groups with 70 percentaccuracy on the basis of the changes in the number of copiesof genomic DNA. In two-way comparisons, melanomas arising onskin with signs of chronic sun-induced damage and skin withoutsuch signs could be correctly classified with 84 percent accuracy.Acral melanoma could be distinguished from mucosal melanomawith 89 percent accuracy. Eighty-one percent of melanomas onskin without chronic sun-induced damage had mutations in BRAFor N-RAS; the majority of melanomas in the other groups hadmutations in neither gene. Melanomas with wild-type BRAF orN-RAS frequently had increases in the number of copies of thegenes for cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1),downstream components of the RAS–BRAF pathway.
Conclusions The genetic alterations identified in melanomasat different sites and with different levels of sun exposureindicate that there are distinct genetic pathways in the developmentof melanoma and implicate CDK4 and CCND1 as independent oncogenesin melanomas without mutations in BRAF or N-RAS.
The rising incidence of melanoma and lack of effective treatmentsfor advanced disease represent an important public health problem.1Exposure to the sun is generally accepted as a major causativefactor.1,2,3 However, its mechanism is unknown, and the roleof exposure to ultraviolet light is complex and has some paradoxicalfeatures. For example, in light-skinned people, the group thatis predominantly affected by melanoma, tumors are most commonon areas that are intermittently exposed to the sun, such asthe trunk, arms, and legs, rather than on areas that are chronicallyexposed to the sun, such as the face. Also, several studieshave shown that indoor workers have a higher risk of melanomathan outdoor workers,4,5 leading some authorities to suggestthat chronic exposure to ultraviolet light exerts a protectiveeffect. A small proportion of melanomas arise without obviousexposure to ultraviolet light, because they affect sites thatare relatively or absolutely protected, such as the palms andsoles (acral melanoma) and mucosal membranes. Finally, genessuch as BRAF and N-RAS that are commonly mutated in melanomado not show typical ultraviolet "fingerprint" mutations.6,7
There has been an ongoing debate about whether this complexitycould in part be due to the existence of several distinct typesof melanoma. One proposal, based on histologic growth patterns,describes four "histogenetic" types of melanoma: superficialspreading, lentigo maligna, nodular, and acral lentiginous melanoma.8,9However, the use of this classification is controversial10 andhas not been broadly adopted in clinical practice, primarilybecause a substantial number of melanomas do not fit the classictypes and the histogenetic type is not an independent prognosticfactor.11,12 A more recent hypothesis suggests that these tumorsbe classified according to divergent pathways, because patientswith melanomas of the head and neck differ from patients withmelanomas on the trunk in having higher levels of expressionof TP53 protein, a higher frequency of associated nonmelanomaskin cancers, and lower numbers of melanocytic nevi.13,14,15
Genetic studies have provided support for this dual-pathwayhypothesis concerning melanomas on skin exposed to the sun.BRAF mutations are common only in melanomas arising in areasintermittently exposed to the sun and are rare in melanomason skin that is chronically exposed to the sun or on acral skinand mucosal membranes that are seldom or never exposed to thesun.16 In addition, several studies have shown that melanomasof the palms and soles and mucosal membranes have distinctivepatterns of chromosomal aberrations as compared with those atother sites.17,18,19 Understanding whether the heterogeneityof melanoma with respect to the site, degree of exposure tothe sun, and histologic characteristics is caused by biologicallydistinct types of melanoma is of great clinical importance,because it is likely to result in separate targeted therapeuticapproaches and prevention strategies. To shed light on thisarea, we analyzed 126 primary melanomas classified into fourgroups on the basis of their location and degree of exposureto the sun. Our analysis included a genome-wide assessment ofthe differences in the number of copies of DNA that used array-basedcomparative genomic hybridization20,21 and a focused analysisof signaling pathways that are markedly altered in melanoma(Figure 1).
Figure 1. The Mitogen-Activated Protein (MAP) Kinase and Phosphatidylinositol 3' Kinase (PI3K) Pathways.
Signals from receptor tyrosine kinases can promote proliferation through the MAP kinase pathway (left branch) and survival through the PI3 kinase pathway (right branch).
Methods
Tumor Specimens
We collected archival, paraffin-embedded primary melanomas thathad an invasive component in which tumor cells predominatedover stromal cells from seven centers: the DermatopathologySection of the Department of Pathology and Dermatology, Universityof California, San Francisco; the Department of Dermatology,Kumamoto University School of Medicine, Kumamoto, Japan; theDepartment of Pathology, Memorial Sloan-Kettering Cancer Center,New York; DermPath, Friedrichshafen, Germany; the Departmentof Dermatology, Seoul National University College of Medicine,Seoul, South Korea; the Department of Dermatology, Tohoku UniversityGraduate School of Medicine, Sendai, Japan; and the Departmentof Dermatology, University of Würzburg, Würzburg,Germany. The study was approved by the institutional reviewboard of the University of California, San Francisco. We obtainedroughly similar numbers of four types of tumors: 36 specimensof acral melanoma, defined as melanoma occurring on the non–hair-bearingskin of the palms or soles or under the nails; 20 specimensof mucosal melanoma, defined as tumors arising on mucosal membranes;30 specimens of melanoma arising from skin with chronic sun-induceddamage; and 40 specimens of melanoma arising from skin withoutchronic sun-induced damage. The distinction between the lasttwo groups was based solely on the presence or absence on microscopyof marked solar elastosis of the dermis surrounding the melanomas.In all but a few cases, melanomas associated with chronic sun-induceddamage occurred on the face and melanomas that were not associatedwith chronic sun-induced damage occurred on the trunk, arms,and legs (Table 1; further details are provided in the Supplementary Appendix,available with the full text of this article at www.nejm.org).
Table 1. Characteristics of the Four Types of Melanoma.
Experimental Methods
DNA for comparative genomic hybridization was extracted fromtumor-bearing tissue as described previously.22 Array-basedcomparative genomic hybridization was carried out on 600 to2000 ng of genomic DNA, labeled by random priming, as previouslydescribed.23 Data points of low quality, as assessed by a largestandard deviation between replicate spots on each array, wererejected. Clones that had missing data in more than 25 percentof the samples in the group as a whole or in 50 percent in anyindividual group were excluded from further analysis (the Supplementary Appendixprovides details of primary data processing). The dataset used for comparative genomic hybridization has been depositedin the Gene Expression Omnibus (accession number, GSE2631
[NCBI GEO]
; availableat www.ncbi.nlm.nih.gov/geo).
Immunohistochemical analysis was performed on tissue microarraysas described previously with the use of standard protocols andthe use of 3-amino-9-ethylcarbazole as a chromagen accordingto the manufacturer's specifications.24 The following antibodieswere used: monoclonal antibody AM29 against cyclin D1 (CCND1;catalog number, 18-0220; Zymed) in a 1:200 dilution, as describedpreviously25; monoclonal antibody Ab-4 against cyclin-dependentkinase N2A (CDKN2A; catalog number, MS-887-P1; Laboratory Vision)in a 1:25 dilution; a polyclonal antibody against phosphorylatedAkt (Ser473; catalog number, 9277S; Cell Signaling Technology)in a 1:80 dilution; and E10 monoclonal antibody against phosphorylatedextracellular-signal–regulated kinase (ERK, Thr202/Tyr204;catalog number, 9106S; Cell Signaling Technology) in a 1:80dilution. The intensity of staining was scored on a scale onwhich a score of 0 indicated the lowest intensity and a scoreof 4 the highest intensity. Scores of 2 or greater were consideredpositive.
We used a polymerase-chain-reaction (PCR) assay to amplify DNAthat included BRAF codon 600 (113 specimens) and codons 12,13, and 61 of N-RAS (113 specimens) and H-RAS and K-RAS (95specimens each). PCR products were purified with the use ofExoSAP-IT (USB), sequenced directly with the use of specificprimers, and analyzed with the use of an ABI Prism 3700 DNAAnalyzer (Applied Biosystems).
Statistical Analysis
The experimental variability of each sample included in thearray for comparative genomic hybridization was assessed asdescribed previously.26 For each hybridization, the median absolutedeviation, a robust estimate of the standard deviation, wascalculated for each portion of the genome found to have a constantnumber of copies, and the median of those values was used toestimate the noise level of the measurement. Changes in thenumbers of copies of DNA within chromosomes and of entire chromosomeswere identified by applying the unsupervised hidden-Markov-modelprocedure as previously described26,27 and used to assess overallgenomic instability.23 Microarray elements with absolute base-2logarithm (log2) ratios greater than 2.5 times the median absolutedeviation were classified as aberrant (having gains or lossesin numbers of copies). The proportion of the genome alteredin each sample was computed as the proportion of aberrant clones.Genomic regions represented by microarray elements were declaredto be homozygously lost if their log2 ratio was less than –0.9.The height of the peak representing the increase in the numberof copies and the narrowness of the affected region relativeto its flanking segments were used to define high-level amplification(details are provided in the Supplementary Appendix). Ampliconswere defined as contiguous regions of amplification. Missingvalues were imputed with the use of the lowess approach, whichis a robust, locally linear regression method.28 The F statistic,corrected for multiple testing with the use of maxT-adjustedpermutation-based P values (i.e., adjusted for multiple testingin a way that strongly controls the probability of declaringfalse positives), was used to assess the significance of differencesbetween the ratios of individual clones among the four groupsof melanomas.29 Adjusted P values of less than 0.05 were consideredto indicate statistical significance, ensuring the probabilityof at least one false discovery of no more than 0.05 and thusproviding strong control of the family-wise error rate.29
We compared individual clones containing genes of interest betweentwo or more groups using the Kruskal–Wallis test. We usedFisher's exact test to evaluate the two-by-two tables comparingmutational status or cluster assignments among the four groups.The rate of misclassification of the classifiers was assessedwith the use of the leave-one-out cross-validation method byrepeatedly applying a diagonal linear discriminant analysisclassifier while varying the number of features used by thepredictor.30 Features were reselected at each cross-validationstep with the use of F-statistic ranking. Hence, only samplesin the most current training set were used, thus eliminatingpotential downward bias of the resulting error rate.30 The reportedleave-one-out error rates correspond to the predictor with thenumber of features resulting in the best performance of theclassifier. All calculations were done in R statistical language.31
Results
Distinct Sets of Genomic Aberrations among Melanoma Subgroups
Array-based comparative genomic hybridization showed that severalgenomic regions had changes in the number of copies of DNA inall four groups of melanoma (Figure 2). In addition, there weremarked differences in aberrant genomic regions among the groups(Figure 2 and Table 1). These differences were most pronouncedbetween melanomas on skin that was relatively or absolutelyprotected from the sun (acral and mucosal melanomas) and melanomason skin with various degrees of exposure to the sun. Specifically,acral or mucosal melanomas had a significantly higher degreeof chromosomal aberrations, as assessed by the overall proportionof the genome affected by gains or losses of DNA (P=0.004 bythe Kruskal–Wallis test), changes in the total numberof copy-number transitions within chromosomes (P<0.001 bythe Kruskal–Wallis test), and changes in the number ofamplicons (P<0.001 by the Kruskal–Wallis test) (Figure 3).Amplifications were found in 89 percent of acral melanomasand 85 percent of mucosal melanomas, but they involved differentgenomic regions in the two groups (Figure 2 and Table 1). Amplificationswere infrequent in the group of melanomas on skin with chronicsun-induced damage and the group on skin without chronic sun-induceddamage. In addition to these differences in the degree of genomicinstability, there were differences in aberration patterns amongthe four groups (Figure 2).
Figure 2. Changes in the Number of Copies of DNA in Subgroups of Melanoma.
Two histograms are shown for each group of melanoma (Panels A, B, C, and D) and for the four groups combined (Panel E). In each panel, the upper plots show low-level gains (green) and losses (red), and the lower plots show amplifications (green) and homozygous deletions (red) for each type. Vertical dashed lines represent the location of the centromere. The x axis represents genomic position, with the bacterial artificial chromosomes ordered according to position in the genome beginning at 1p and ending at 22. The y axis represents the fraction of the samples with a given clone altered. Panel E shows the statistical differences between the changes in the number of copies in the four groups. The magnitude of the F statistic is shown as the height of the vertical bars, and their global significance is indicated with horizontal dashed lines that show the maxT-adjusted P-value cutoffs (red, P=0.01; blue, P=0.05; and green, P=0.1).
Figure 3. Qualitative and Quantitative Differences in Chromosomal Aberrations among the Four Groups of Melanoma.
The box plots indicate significant differences in the number of copies of DNA (Panel A) and number of amplicons (Panel B) among the four types of melanoma (P<0.001 by the Kruskal–Wallis test). Boxes are outlined by the first and third quartiles, with the median as a horizontal line inside. Whiskers are drawn as 1.5 times the distance between the first and third quartiles. Points outside the whiskers are considered outliers. The notches indicate the 95 percent confidence interval for the median. Panel C shows the unsupervised hierarchical clustering of melanomas on skin with chronic sun-induced damage and melanomas on skin without chronic sun-induced damage. Genomic clones are displayed in vertical orientation according to their genomic position from chromosomes 1 to 22. The p arms of chromosomes are indicated in light blue (odd chromosomes) and yellow (even chromosomes), and the q arms are indicated in dark blue (odd chromosomes) and green (even chromosomes). The CCND1 locus was frequently gained in the right branch of the tree (arrowhead), which was enriched for specimens from skin with chronic sun-induced damage (P=0.001 by the Kruskal–Wallis test). Ten samples from skin without chronic sun-induced damage were incorrectly grouped with samples from skin with such damage, and 10 samples from skin with chronic sun-induced damage were incorrectly grouped with samples from skin without such damage. There was a significant association between groups and cluster assignment (P<0.001 by Fisher's exact test).
Table 1 shows the common genomic aberrations and the regionsthat were affected with different frequencies in each groupafter adjustment for multiple testing. On the basis of the changesin the number of copies of DNA alone, samples could be correctlyclassified into the four groups with an overall accuracy of70 percent. Two-way classification of acral and mucosal melanomascorrectly classified 50 of 56 samples (89 percent). Most intriguingly,two-way classification of the group of melanomas on skin withchronic sun-induced damage and the group on skin without chronicsun-induced damage led to the correct classification of 59 of70 samples (84 percent). Unsupervised agglomerative hierarchicalclustering separated the group of melanomas that were on skinwith chronic sun-induced damage and the group on skin withoutchronic sun-induced damage that had been exposed to sun intotwo major clusters (P<0.001 by Fisher's exact test) (Figure 3C).Frequent focused gains involving the CCND1 locus (P=0.001),losses involving chromosome 4q (P=0.004), and gains involvingregions of chromosome 22 (P=0.004) were significantly more commonin the group with chronic sun-induced damage than in the groupwithout such damage; the latter group had more frequent lossesinvolving chromosome 10q (P=0.002). All P values were calculatedwith the use of a maxT-adjusted permutation-based t-test asdescribed above and are reported for the most significant changesof the relevant chromosomal regions.
Alteration of the Mitogen-Activated Protein Kinase Pathway
Mutations in BRAF were significantly more common in the groupof melanomas that were on skin without chronic sun-induced damagethan in the other three groups (P<0.001 by Fisher's exacttest) (Figure 4F), confirming our previous report.16 All mutationsin RAS genes were found in N-RAS and occurred only in sampleswithout BRAF mutations. We did not observe a significant associationbetween N-RAS mutations and melanoma subtypes (P=0.4 by Fisher'sexact test) (Figure 4F).
Figure 4. Genetic Alterations in the Mitogen-Activated Protein Kinase and PI3K Pathways.
Panels A, B, and C show examples of individual specimens with focal amplification of CCND1 (Panel A) and CDK4 (Panel B) and homozygous deletion of CDKN2A (Panel C). The y axis represents the mean log2 ratio for each clone normalized to the genome median log2 ratio. The x axis represents individual clones ordered with the use of data obtained from the University of California at Santa Cruz Genome Browser (http://genome.ucsc.edu, assembly of July 2003). Panels D and E show interactions between amplification of CDK4 and CCND1 and mutations in BRAF and N-RAS (Panel D) and homozygous deletions of CDKN2A and amplifications of CDK4 (Panel E). The x and y axes show the log2 ratio for the clone representing the individual genes. Data points labeled in red or blue indicate cases with mutations in the BRAF or N-RAS genes, respectively. The average log2 ratio of three highly correlated clones (minimum Pearson's correlation, >0.95) containing the CCND1 gene were used in Panels D and G. Panel F shows a significant difference in the mutation frequency of BRAF but not N-RAS among the four groups. Panel G illustrates significant differences in the numbers of copies of CDKN2A, PTEN, CCND1, and CDK4 among the four types of melanoma. Boxes are outlined by the first and third quartiles, with the median as a horizontal line inside. Whiskers are drawn as 1.5 times the distance between the first and third quartiles. Points outside the whiskers are considered outliers. In Panel G, enh denotes an increased number of copies, dim a reduced number of copies, amp amplification, and hom del homozygous deletion. The y axis indicates the log2 ratio of the clone representative of the respective gene. CSD denotes chronic sun-induced damage, and non-CSD no chronic sun-induced damage.
Immunohistochemical analysis showed that samples without mutationsin BRAF or N-RAS less frequently expressed phosphorylated ERKthan did samples with such mutations, suggesting that in sampleswith wild-type BRAF and N-RAS, the pathway was not activatedupstream of phosphorylated ERK (see the Supplementary Appendix).One downstream gene, CCND1, resided in the most common genomicregion affected by focused amplifications (Figure 4A). The increasesin the number of copies of CCND1 were inversely correlated withmutations in BRAF (P=0.008 by the Kruskal–Wallis test),independently of the type of melanoma (Figure 4D). Specimensthat had increased levels of expression of CCND1 on immunohistochemicalanalysis had without exception either mutations in BRAF or N-RASor an increased number of copies of CCND1 (see the Supplementary Appendix).This observation and the strong inverse correlationbetween BRAF mutations and the increase in the number of copiesof CCND1 suggest that the elevation in the levels of CCND1 proteinas a result of either mutations in upstream genes or increasesin the gene dosage represents a crucial event driving progressionin melanoma.
Interestingly, CDK4, whose protein is one of the binding partnersof CCND1 and which is located on chromosome 12q14, was alsosubject to recurrent focal amplifications (Figure 4B). CDK4amplifications were more common in acral and mucosal melanomasthan in the other two groups (P=0.005 by the Kruskal–Wallistest). None of the 11 specimens with CDK4 amplifications hadmutations in either BRAF or N-RAS or had amplification of CCND1(Figure 4D). The strong complementarity of the genetic interactionsamong CDK4, CCND1, BRAF, and N-RAS implicate CDK4 and CCND1as independent oncogenes in melanoma.
The CDKN2A protein (also commonly known as p16) is a melanoma-susceptibilityfactor and the main negative regulator of the CDK4–CCND1complex, representing a crucial gatekeeper at the G1–Scheckpoint.32 We found deletions of the CDKN2A locus in 50 percentof all melanomas, making it the most commonly lost genomic region(Figure 4G). In 10 percent of our specimens, comparative genomichybridization showed a homozygous deletion of the CDKN2A locusparalleled by a complete loss of expression of CDKN2A proteinon immunohistochemical analysis (see the Supplementary Appendix).Mucosal and acral melanomas more commonly had losses of theCDKN2A locus than did the group of melanomas on skin with chronicsun-induced damage or the group on skin without chronic sun-induceddamage (P<0.001 by the Kruskal–Wallis test). Homozygousdeletions of CDKN2A occurred exclusively in samples withoutCDK4 amplifications (Figure 4E). This observation suggests thatamplification of CDK4 obviates the need for deletions of CDKN2A.Since the CDKN2A and CDK4 proteins bind each other in a 1:1ratio,33 the evolution of melanoma appears to be enhanced bya decrease in the ratio of CDKN2A to CDK4 that loosens checkpointcontrol for entry into the S phase. We did not note an influenceof the tumor thickness on either the frequencies of mutationsin BRAF or N-RAS or on the amplification frequencies of CCND1or CDK4 in the four groups of melanomas.
Alterations of the Phosphatidylinositol 3' Kinase Pathway
Several studies have implicated activation of the phosphatidylinositol3' kinase (PI3K) pathway as another crucial event in the progressionof melanoma. To date, PTEN, a negative regulator of the pathway,appears to be the most common somatic target in melanoma, residingin a commonly deleted genomic area on chromosome 10q. In melanomacell lines, mutations or deletions of PTEN occur together withmutations in BRAF, but not in N-RAS.34 N-RAS activates boththe PI3K pathway and the mitogen-activated protein kinase pathway,whereas BRAF seems to activate only the latter, suggesting thatduring progression, somatic events activating only one pathwayrequire a second event to activate the other. Our data are consistentwith this finding. Specimens with BRAF mutations had fewer copiesof PTEN than specimens with N-RAS mutations (P=0.02 by the Kruskal–Wallistest). We confirmed that the PI3K pathway had been activatedby evaluating 51 specimens with sufficient tissue for immunohistochemicalanalysis of the expression of phosphorylated Akt. In this subgroupof samples, all 8 specimens with N-RAS mutations had increasedlevels of expression of phosphorylated Akt, and 14 of 17 specimens(82 percent) with PTEN loss had increased levels of expressionof phosphorylated Akt (see the Supplementary Appendix). Thepositive correlation between the loss of PTEN and mutationsin BRAF supports the notion that the PI3K pathway is an independentsomatic target that is frequently activated in primary melanoma.
Discussion
Our study shows distinct patterns of genetic alterations inthe four groups of primary melanomas as we have defined them.There were differences in both chromosomal aberrations and thefrequency of mutations of specific genes, suggesting that thesetumors develop by different mechanistic routes in response todifferent selective influences. For example, acral and mucosalmelanomas were uniquely characterized by a much higher frequencyof focal amplifications and losses than the group of melanomasthat were on skin with chronic sun-induced damage or the groupon skin without chronic sun-induced damage, indicating thatthe first two are mechanistically more similar to each otherthan to the other types. However, as our data show, selectionresults in highly divergent sets of chromosomal aberrationsin acral and mucosal tumors, findings that support their classificationas distinct entities. Because glabrous and mucosal epitheliaare morphologically distinct from each other and because solarexposure is unlikely to contribute to melanomagenesis in thesesites, we believe that these two groups of melanomas differprimarily as the result of site-specific biologic characteristics.We also found clear genetic differences between the types ofmelanoma that commonly occur on areas exposed to the sun inlight-skinned people. Melanomas on skin without chronic sun-induceddamage had frequent mutations in BRAF and frequent losses ofchromosome 10, whereas melanomas on skin with chronic sun-induceddamage had infrequent mutations in BRAF and frequent increasesin the number of copies of the CCND1 gene.
Our classification of melanomas related to exposure to ultravioletlight is based on examination of our data and data from previousepidemiologic studies.14 The previous studies found that variousrisk factors differed between melanomas on skin with chronicsun-induced damage and melanomas on skin without chronic sun-induceddamage and suggested the existence of different developmentalpaths for these types of tumors. For example, melanomas presentingon sites with chronic exposure to the sun typically occur latein life and are associated with other ultraviolet-light–relatedneoplasms such as solar keratoses, suggesting that high cumulativedoses of ultraviolet light are required for their development.In contrast, melanomas presenting on skin that is intermittentlyexposed to the sun are typically found in persons who have alarger number of moles but fewer solar keratoses and occur ata younger age. Moreover, these melanomas and the moles sharean important genetic characteristic. In this and a previousstudy,16 we have shown that the melanomas on skin that is intermittentlyexposed to the sun have much more frequent mutations of BRAFthan do other types of melanoma, and moles have also been shownto have a high incidence of BRAF mutations.35 Thus, we proposethat the melanocytes of persons in whom melanomas develop onskin that is intermittently exposed to the sun have an increasedsusceptibility to ultraviolet exposure that involves a higherprobability of acquiring BRAF mutations or of proliferatingif such mutations occur. Epidemiologic studies and studies inanimals indicate that for these people, there may be a windowof vulnerability to exposure to ultraviolet light early in life.2,36,37These tumors should occur with site-specific incidences relatedto the patterns and timing of exposure to ultraviolet lightand the relative amount of skin at risk. In contrast, patientswithout susceptible melanocytes would require a sufficientlyhigh cumulative ultraviolet dose to induce melanoma, so thatsun-damaged skin would be present and the development wouldinvolve mechanisms that do not include BRAF mutations. Thus,these persons would have tumors predominantly on sites thatare chronically exposed to the sun, such as the face.
Although we have clearly established that there are geneticdifferences among these four groups of melanomas, our classificationrequires validation and refinement. Three important questionscome immediately to mind. First, in distinguishing between acraland mucosal melanomas, we have emphasized differences in theaffected site and morphologic differences in skin but not thelimited exposure to ultraviolet light that acral sites may receive.Second, for sites subject to substantial exposure to ultravioletlight, we have emphasized the importance of histologic signsof sun-induced damage and have not considered the possibilityproposed by Whiteman et al.14 that there could be differencesin susceptibility between melanocytes found on the trunk, arms,or legs and those found on the face. Third, we discounted thehistologic tumor type because we found that tumors on acralsites have similar genetic aberrations regardless of whetherthey are histologically acral lentiginous or superficial spreadingmelanomas and that nodular melanomas clustered with the groupswe defined on the basis of site and did not have the featuresof a separate entity. Future epidemiologic and genetic studieswill be required to identify specific genetic or epigeneticfactors associated with these variables.
Knowledge of the genetic differences among melanomas could bevaluable in the design of therapeutic strategies. Our resultslead us to make a prediction. The group of tumors on skin withoutchronic sun-induced damage, which represent the most commontype of melanoma, frequently had a mutation in BRAF togetherwith a loss of PTEN or mutations in N-RAS alone. Thus, theywould be expected to be responsive to therapeutic interventionstargeting the RAS–RAF–ERK and PI3K pathways. Incontrast, the majority of melanomas in the other three groupsdid not have mutations in BRAF or N-RAS but instead had increasednumbers of copies of the downstream gene CCND1 or CDK4. Thus,these three groups of melanomas would be less likely to respondto therapeutic interventions that target upstream componentsof the mitogen-activated protein kinase pathway including BRAF,such as sorafenib.38
Our study provides genetic support for the existence of distinctmolecular pathways to melanoma, each with a unique relationshipto exposure to ultraviolet light. This finding should affectthe design of future studies involving the treatment and preventionof melanoma and suggests the existence of as-yet-unidentifiedsusceptibility factors.
Supported by grants (R33 CA95300 and R01 CA094963) from theNational Cancer Institute.
We are indebted to Drs. John L. Ziegler and Ervin H. Epstein,Jr., for helpful comments on the manuscript and to Susan Charzanand Dr. George Carter for their technical assistance.
Source Information
From the Comprehensive Cancer Center (J.A.C., J.F., H.N.P., D.P., B.C.B.) and the Departments of Epidemiology and Biostatistics (J.F.) and Dermatology and Pathology (P.E.L., B.C.B.), University of California, San Francisco, San Francisco; the Department of Dermatology, Kumamoto University School of Medicine, Kumamoto, Japan (T.K.); the Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York (K.J.B.); DermPath, Friedrichshafen, Germany (H.K.); the Department of Dermatology, Seoul National University College of Medicine, Seoul, South Korea (K.-H.C.); the Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan (S.A.); and the Department of Dermatology, University of Würzburg, Würzburg, Germany (E.-B.B.).
Address reprint requests to Dr. Bastian at UCSF Cancer Center, Box 0808, San Francisco, CA 94143-0808, or at bastian{at}cc.ucsf.edu.
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