Originally published as JCO Early Release 10.1200/JCO.2006.06.2984 on August 14 2006

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Somatic Activation of KIT in Distinct Subtypes of Melanoma

John A. Curtin, Klaus Busam, Daniel Pinkel, Boris C. Bastian

From the Comprehensive Cancer Center; Departments of Laboratory Medicine, Dermatology, and Pathology, University of California, San Francisco, San Francisco, CA; and the Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY

Address reprint requests to Boris C. Bastian, MD, UCSF Cancer Center, Box 0808, San Francisco, CA 94143-0808; e-mail: bastian{at}cc.ucsf.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Purpose Melanomas on mucosal membranes, acral skin (soles, palms, and nail bed), and skin with chronic sun-induced damage have infrequent mutations in BRAF and NRAS, genes within the mitogen-activated protein (MAP) kinase pathway commonly mutated in melanomas on intermittently sun-exposed skin. This raises the question of whether other aberrations are occurring in the MAP kinase cascade in the melanoma types with infrequent mutations of BRAF and NRAS.

Patients and Methods We analyzed array comparative genomic hybridization data from 102 primary melanomas (38 from mucosa, 28 from acral skin, and 18 from skin with and 18 from skin without chronic sun-induced damage) for DNA copy number aberrations specific to melanoma subtypes where mutations in BRAF and NRAS are infrequent. A narrow amplification on 4q12 was found, and candidate genes within it were analyzed.

Results Oncogenic mutations in KIT were found in three of seven tumors with amplifications. Examination of all 102 primary melanomas found mutations and/or copy number increases of KIT in 39% of mucosal, 36% of acral, and 28% of melanomas on chronically sun-damaged skin, but not in any (0%) melanomas on skin without chronic sun damage. Seventy-nine percent of tumors with mutations and 53% of tumors with multiple copies of KIT demonstrated increased KIT protein levels.

Conclusion KIT is an important oncogene in melanoma. Because the majority of the KIT mutations we found in melanoma also occur in imatinib-responsive cancers of other types, imatinib may offer an immediate therapeutic benefit for a significant proportion of the global melanoma burden.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Although abnormalities in a limited number of interacting regulatory networks appear to be involved in cancer development, the many components of these networks offer numerous possibilities for disruption in a particular tumor. In some cases, the specific site of abnormality depends on tumor type or subtype. As we have recently shown in melanoma,¹ the mitogen-activated protein (MAP) kinase and phosphatidylinositol 3 (PI3) kinase pathways are activated differently among subtypes of melanoma when tumors are classified into four groups according to a combination of sun exposure and anatomic site. Most prominently, although BRAF mutations are highly prevalent (59%) in melanomas occurring on skin without signs of chronic sun-induced damage (non-CSD melanomas), BRAF mutations occur significantly less frequent in melanomas on sun-protected skin such as the palms, soles, or subungual sites (acral melanomas), and on mucosal membranes (mucosal melanomas). BRAF mutations are also uncommon in melanomas that occur on skin showing evidence of chronic sun-induced damage evidenced by marked solar elastosis (CSD melanomas). Approximately 10% to 20% of melanomas of all four subtypes activate the MAP kinase pathway by mutation of NRAS, but mutations of both NRAS and BRAF virtually never occur together. These findings raised the critical question of how this pathway might be activated in those tumors that do not have NRAS or BRAF mutations.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Study Population
We studied 102 primary melanomas from mucosa (n = 38), acral skin (n = 28), and skin with (n = 18) and without (n = 18) chronic sun-induced damage. Sun-induced damage was defined microscopically by the presence or absence of marked solar elastosis. Eighty cases were part of a previously published data set.^1,2 The additional 22 samples, all mucosal melanomas, were obtained from the archives of the Department of Pathology, University of California, San Francisco (San Francisco, CA) and the Department of Pathology, Memorial Sloan-Kettering Cancer Center (New York, NY). The study was approved by the institutional review board of the University of California, San Francisco.

Experimental Methods
DNA for comparative genomic hybridization (CGH) and mutation analysis was extracted from formalin-fixed, paraffin-embedded primary tumors and surrounding healthy tissue as published previously.³ Array CGH hybridization on samples not previously analyzed¹ was carried out on 600 to 2,000 ng of genomic DNA, labeled by random priming, and analyzed as described previously.^1,4 The definition of gains and losses are described in detail elsewhere.¹ Briefly, an amplicon is defined as a focal (narrower than 10 Mbases) copy number increase whose log₂ ratios exceed immediately flanking segments by 0.9, and a gain is defined as a copy number increase that exceeds a log₂ ratio of 0.35 but does not fulfill the definition of amplification.

Immunohistochemistry was performed on tissue microarrays using standard protocols with 3-amino-9-ethylcarbazole as a chromogen following the manufacturer's specifications as previously described,⁵ as well as on tissue sections. We used the polyclonal rabbit antihuman KIT antibody CD117 (Dako Cytomation Inc, Carpinteria, CA) at a dilution of 1:200. In tumors with mutations or increased copies of KIT that did not show positive staining in their invasive portions at the 1:200 dilution, a dilution of 1:25 was used to determine whether the protein was expressed at lower levels. Staining intensity levels were scored from 0 to 4 where 0 is the lowest and 4 the highest intensity. Intensity levels of 2 or greater were considered positive. A sample with known positive staining was used as an external positive control and expression of the protein in mast cells present within the skin surrounding the melanomas as an internal positive control. Lack of protein staining in the epidermis served as a negative control.

We performed sequence analysis by direct sequencing of polymerase chain reaction (PCR) –amplified products generated with specific primers designed to include the exons of interest. The primers used were as follows: exon 11, 5'-TGTTCTCTCTCCAGAGTGCTCTAA-3' (forward) and 5'-AAACAAAGGAAGCCACTGGA-3' (reverse); exon 13, 5'-CATCAGTTTGCCAGTTGTGC-3' (forward) and 5'-AGCAAGAGAGAACAACAGTCTGG-3' (reverse); exon 17, 5'-TCATTCAAGGCGTACTTTTG-3' (forward) and 5'-TCGAAAGTTGAAACTAAAAATCC-3' (reverse); and exon 18, 5'-CATTTCAGCAACAGCAGCAT-3' (forward) and 5'-CAAGGAAGCAGGACACCAAT-3' (reverse). For sequencing purposes, an M13 forward (5'-TGTAAAACGACGGCCAGT-3') and reverse (5'-AGCGGATAACAATTTCACACAGG-3') primer was added onto the 5' terminals of each forward and reverse primer respectively. The PCR cycling conditions were as follows. An initial denaturation at 95°C for 5 minutes was followed by eight cycles of 95°C for 30 seconds, an annealing phase at 61°C for 90 seconds decreasing by 0.5°C every cycle to touch down at 57°C, and an elongation step of 72°C for 90 seconds. This was followed by an additional 30 cycles as described herein with an annealing temperature of 57°C, and finally an elongation step at 72°C for 10 minutes. PCR products were purified using ExoSAP-IT (USB Corporation, Cleveland, OH) and sequenced directly with M13 primers using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, CA).

Statistical Methods
The two-tailed Mann-Whitney U test was used to compare KIT expression levels between melanomas with KIT mutations or copy number increases to melanomas without such aberrations. P values less than .05 were regarded as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Examination of the array CGH copy number profiles in a cohort of 102 primary melanomas that contained approximately equal representation of the four melanoma subtypes found seven tumors with overlapping amplifications on chromosome 4q12 (Fig 1) and an additional 11 with increased copy number affecting that region. All 18 tumors were of the acral, mucosal, or CSD subtypes. Seventeen of these tumors had been sequenced for BRAF and NRAS, and no mutations were found. All array CGH data have been deposited in the Gene Expression Omnibus available at www.ncbi.nlm.nih.gov/geo/ under the accession numbers listed in Table A1 (online only).

View larger version (41K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. KIT alterations. (Left panels) DNA copy number profiles (log 2 ratio v genomic position) for two tumors, (A) and (B). Red arrows indicate KIT amplifications. (Middle panels) Sequencing shows mutations corresponding to K642E amino acid substitutions. (Right panels) KIT protein overexpression in invading portions of both tumors, including cells in lymphatics (black arrows) in tumor (B).

Immunohistochemistry for PDGFRA and KDR and in situ hybridization for PDGFRA on tissue microarrays showed that although both genes were expressed in some melanomas, there was no association between copy number increase at 4q12 and protein or RNA expression (data not shown). In addition, sequencing of the common mutation sites of PDGFRA, exons 10, 12, 14 and 18,²¹ in cases with amplification of 4q12 did not reveal any mutations. Tumors without amplifications were not sequenced, and so we cannot rule out the possibility that PDGFRA mutations may be present in some of these tumors. In contrast, sequence analysis of the common mutation sites of KIT, exons 11, 13, 17, and 18 in the seven samples with amplifications in KIT found three with mutations. All had a K642E mutation, and one had an additional mutation at residue N566D (Table 1). The K642E mutation is oncogenic¹⁰ and occurs in sporadic¹¹ and familial GISTs (Table 2).¹⁰ The amplification at 4q12 targeted the mutated allele in all three melanomas as indicated by the peak height of the sequencing traces, and KIT protein was highly expressed in all three cases (Fig 1). These findings indicated the high likelihood that KIT was the gene driving the selection of copy number increases at 4q12 in at least these three cases, and motivated sequencing these same KIT exons in the remaining 95 of the tumors to determine whether mutations were occurring in the absence of copy number changes. Coding mutations in 10 of these and an intronic deletion in an additional tumor were found (Table 1). Analysis of DNA from adjacent normal tissue for the nine of the total 15 KIT-mutant cases from which it could be obtained, found no mutations, indicating that the mutations were somatically acquired. The frequencies of KIT aberrations (ie, mutations or copy number increases) in the different melanoma subgroups varied in an approximate mirror image with BRAF mutations (Fig 2). KIT aberrations were found in 15 (39%) of 38 of mucosal, 10 (36%) of 28 of acral, five (28%) of 18 on skin with chronic sun-induced damage, and none (0%) of 18 of melanomas on skin without chronic sun-induced damage. By contrast, BRAF mutations were present in 3%, 21%, 6%, and 56%, respectively.

View larger version (18K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Frequency distribution of genetic alterations in BRAF, NRAS, and KIT among four groups of melanoma. Non-CSD, melanomas on skin without chronic sun-induced damage; CSD, melanomas on skin with chronic sun-induced as evidenced by the presence of marked solar elastosis; acral, melanomas on the soles, palms, or sub-ungual sites; mucosal, melanomas on mucosal membranes. One CSD melanoma had a KIT and an NRAS mutation, and one acral melanoma had a KIT and a BRAF mutation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
We have demonstrated that genetic aberrations affecting KIT occur frequently in melanomas on the mucosa, acral skin, and skin with sun-induced damage, but do not occur in melanomas on skin without sun damage. Our findings contrast with most previous studies, which found KIT to be downregulated during melanoma progression, or to negatively affect tumor growth,^13-17 but are not unprecedented. KIT mutations were found in two of 100 metastatic melanomas, but no details about the nature of the primary tumor were provided²³ in this study, and high-level KIT expression was reported in some acral melanomas.²⁴ The discrepancies between our results and the predominant previous literature can be reconciled by the fact that those studies were carried out in melanoma cell lines, which are usually derived from the melanoma subtype characterized by its occurrence on intermittently exposed skin and presence of frequent BRAF mutations.^1,2 The complete absence of mutations or copy number increases of KIT in these cell lines therefore supports the notion that it does not act as an oncogene in this melanoma subtype. Thus, although our melanoma classification scheme is likely to be incomplete, the KIT data demonstrate the value of utilizing it for study design and interpretation, and reinforce its basic validity.

The presence of frequent genetic aberrations affecting KIT has important implications for melanoma therapy, improves the understanding of melanoma development, and suggests future lines of research. Eleven of the melanomas in our series had KIT mutations predicted to affect the juxta-membrane domain (Table 2). These mutations are expected to promote KIT dimerization in the absence of scatter factor (SCF) resulting in its constitutive activation,²⁵ or to prevent KIT from maintaining its auto-inhibited conformation.²⁶ Importantly for therapeutic considerations, other cancer types with mutations in the juxta-membrane region are responsive to imatinib.^27-29 Three cases had mutations in the kinase domain, which are less responsive in other tumor types (Table 2).²⁷ Thus, our results provide a strong motivation for expeditious clinical trials of imatinib for melanoma, and a rational basis for their design and interpretation. We note that although the melanoma types that have the highest prevalence of KIT mutations, those on acral and mucosal skin, are infrequent in European populations compared with melanomas that develop on sun-exposed skin, they are particularly aggressive and have approximately equal incidence in all human populations. Thus these melanoma types represent a substantial proportion of the worldwide melanoma burden, and a considerable therapeutic opportunity. Suggestively, a recent preliminary report of a clinical trial of imatinib in 21 patients with cutaneous melanoma showed one near complete response, which was a patient with stage IV acral melanoma.³⁰ This case was demonstrated to have a deletion affecting a splice site resulting in an aberrant transcript of KIT. A significant proportion of melanomas that present on skin with chronic sun-induced damage in European populations may also respond to imatinib.

The mutation, dosage, and protein expression data indicate the complexities of the manner in which KIT may be involved in aberrant signaling. We observed elevated KIT protein levels in the VGP in 79% of samples with mutation and 53% with multiple copies of KIT under standard antibody conditions, and essentially all of these samples when higher antibody concentrations were employed. Interestingly, 31% of melanomas without detectable KIT mutation or copy number increase also showed increased expression of the KIT protein in the VGP under standard antibody concentrations. Thus, aberrant KIT signaling in melanoma may be activated by mutation, gene dosage effects as described for other cancer types,³¹ or perhaps by other mechanisms not measured in our study. One task of clinical trials with imatinib or possibly other KIT inhibitors will be to determine which combination of mutation, copy number status, or protein expression will best predict clinical response.

Among samples with KIT mutations, only melanomas with the K642E mutation showed copy number increase of KIT or a concomitant BRAF mutation (Table 1). Functional studies³² and the occasional finding of the mutant K642E allele in the germline¹⁰ suggest that it represents a weakly activating form of KIT. Thus, this mutation appears to require additional directly interacting alterations in order to provide a significant oncogenic signal. All other samples with coding mutations in KIT did not have copy number increases of the KIT locus and did not show BRAF or NRAS mutations, suggesting that these mutations have a substantially stronger oncogenic signal.

Melanoma types that have frequent genetic alterations of KIT typically have a lentiginous growth pattern, characterized by melanocytes lined up as single cells along the epidermis in the progression stage preceding invasive growth. By contrast, non-CSD melanomas, which have no KIT mutations or copy number increases, typically show a pagetoid growth pattern with melanocytes scattered throughout the epidermis. Because KIT signaling is involved in melanocyte migration and homing to the basal epidermis, it will be interesting to further analyze the involvement of KIT in the lentiginous growth pattern in melanoma.

In conclusion, our study identifies KIT as an oncogene and potential therapeutic target in melanomas of mucosal membranes and acral skin, and skin with chronic sun-induced damage. These melanoma types only infrequently show mutations in BRAF. The differential roles of KIT among melanoma types provides further evidence for genetically and biologically distinct melanoma types,^1,2,33 emphasizes the need to properly classify these tumors to understand their pathogenesis, and fills in the picture of MAP kinase involvement in melanoma. We expect that alterations affecting additional components of the MAP kinase pathway will be found in those melanomas where none are currently known. Although all of these aberrations may have partial functional equivalence, the clear distinctions in the aberration spectra among the different melanoma types suggests that each may have additional functions that are important for tumor development under particular biologic and environmental conditions.

	Appendix

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Table A1Table A1

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 

Conception and design: Boris C. Bastian Financial support: Boris C. Bastian, Daniel Pinkel Provision of study materials or patients: Klaus Busam, Boris C. Bastian Collection and assembly of data: John A. Curtin Data analysis and interpretation: John A. Curtin, Daniel Pinkel, Boris C. Bastian Manuscript writing: John A. Curtin, Daniel Pinkel, Boris C. Bastian Final approval of manuscript: John A. Curtin, Klaus Busam, Daniel Pinkel, Boris C. Bastian

 

	GLOSSARY

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 

Acral skin:

The non–hair-bearing areas such as soles, palms, and nail beds.

Amplification:

The increase of the copy number of a relatively narrow region of the genome, with the magnitude of the increase being large enough so that its generation requires more than a single aberrant event (eg, typically more than the gain of a single copy of the region.

BRAF:

BRAF is an isoform of RAF. Raf proteins (Raf-1, A-Raf, B-Raf) are intermediate to Ras and MAPK in the cellular proliferative pathway. Raf proteins are typically activated by Ras via phosphorylation, and activated Raf proteins in turn activate MAPK via phosphorylation. However, Raf proteins may also be independently activated by other kinases.

CSD melanoma:

Melanomas with chronic sun-induced damage of the surrounding dermis, as determined by the microscopic presence of marked solar elastosis.

Lentiginous growth pattern:

An intraepidermal growth pattern in which melanocytes are arranged predominantly as single units within the basal layer of the epidermis.

MAP kinase pathway:

Signal transduction pathway important for survival and proliferative signals through the activation of Ras, BRAF, MEK, and ERK.

Non-CSD melanoma:

Melanomas without marked solar elastosis of the surrounding dermis.

NRAS:

An isoform of Ras. The Ras gene family consists of H-Ras, N-Ras, and K-Ras. The Ras proteins are typically small triphosphate-binding proteins, and are the common upstream molecule of several signaling pathways that play a key role in signal transduction, which results in cellular proliferation and transformation.

Pagetoid growth pattern:

An intraepidermal growth pattern, in which melanocytes are scattered through all layers of the epidermis, typically in small clusters, as opposed to remaining within the basal layer.

VGP (vertical growth phase):

A phase of tumor progression in which melanoma cells form a nodule that invades the dermis.

	ACKNOWLEDGMENTS

 
We thank Susan Charzan and Anne Estep for excellent technical assistance and members of the University of California San Francisco Comprehensive Cancer Center Microarray Shared Resource for providing BAC arrays.

	NOTES

 
published online ahead of print at www.jco.org on August 14, 2006.

Suported in part by National Cancer Institute Grants No. R33 CA95300, R01 CA094963, and PO1 CA025874-25-A1.

Presented in part at a National Institutes of Health Resources for Melanoma Research workshop that was not open to the public; and the 97th Annual Meeting of the American Association of Cancer Research, Washington, DC, April 1-5, 2006.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
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Submitted February 22, 2006; accepted May 4, 2006.

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