- Email: [email protected]
Molecular biology of melanoma
Molecular biology of melanoma Julie M. Swick, MD,a and John C. Maize, Sr, MDa,b Charleston, South Carolina Dermatologists and dermatopathologists face the difficulties of accurately diagnosing and treating atypical melanocytic lesions and melanomas. Despite huge advances in medicine, our management of melanoma has not significantly changed in many years. The biggest gains made recently have been in the identification of common mutations in melanoma and the use of these mutations to aid in the diagnosis and treatment of melanoma. To understand these gains one must first be familiar with the regulatory pathways of melanoma and the most common mutations found there. This article will review the function and significance of the most studied mutations in melanoma and briefly discuss new and planned treatment options. ( J Am Acad Dermatol 2012;67:1049-54.) Key words: BRAF; KIT; melanoma; melanoma mutations; mitogen-activated protein kinase; RAS.
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n 2010 there were an estimated 68,130 new cases of melanoma and 8700 deaths from melanoma in the United States.1 At this point in time early detection and excision is our best hope for prolonged survival. The difficulties of melanoma do not stop with awareness and detection but extend to the diagnosis of histologically ambiguous lesions. Accurate microscopic diagnosis of atypical melanocytic lesions is very challenging and discordance in diagnoses has been shown between dermatopathologists, who are experts in the diagnosis of melanocytic neoplasms.2 Great strides have been made in understanding the molecular pathways and mutations from which many melanomas originate. The hope of this research is to identify specific mutations that may be useful as genetic markers to aid in histopathologic diagnosis and targeted mutation-specific treatments.3,4 To understand how individual mutations can be used for diagnosis and treatment we must first review the most commonly mutated genes and genetic pathways.
KIT Key point d
KIT mutations are found in acral and mucosal melanomas, and in melanomas on chronically sun-damaged skin.
From the Department of Dermatology, Medical University of South Carolina,a and DermPath Diagnostics of South Carolina.b Funding sources: None. Conflicts of interest: None declared. Accepted for publication June 7, 2011.
Abbreviations used: CDK: MAPK: PI3K: PTEN:
cyclin-dependent kinase mitogen-activated protein kinase phosphatidylinositol 3’-kinase phosphatase with tensin homolog
The KIT gene encodes a tyrosine kinase membrane receptor that stimulates the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3’-kinase (PI3K) pathways (Fig 1). This gene has long been known to be involved in melanocyte development because of the lack of migration and disappearance of KIT-deficient melanoblasts, as well as the relationship of KIT with melanocytes seen in piebaldism. Studies have shown that KIT activation stimulates melanocyte migration more than proliferation.5 While studying acral melanomas, researchers have found melanocytes with similar chromosomal mutations, but appropriate numbers and spacing, up to 2 cm beyond the melanoma itself.5 Studies have also found a common location for mutation in acral melanomas using molecular genetics in chromosome 4q. This location includes the KIT gene.5 Researchers then began to look for KIT mutations in melanoma using molecular technology techniques and found them present in 11% of acral, 21% of mucosal, and 17% of melanomas appearing
Reprint requests: Julie M. Swick, MD, Department of Dermatology, Medical University of South Carolina, 135 Rutledge Ave, 11th Floor, Charleston, SC 29425. E-mail: [email protected]. Published online March 29, 2012. 0190-9622/$36.00 Ó 2012 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2011.06.047
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on chronically sun-damaged sites. This was in comparison with nonchronic sun-damaged melanomas that often had B-RAF mutations.5
K-RAS mutations have been found in 2% of melanomas. Although this protein plays a very active role in other malignancies, such as pancreatic and colon cancers, the same has not been true for melanoma.11 MAPK PATHWAY H-RAS mutations are present in 1% of melanomas. Key points Mouse models have shown that H-RAS-mutated mice d The MAPK pathway transfers extracellular require a loss of p16, p53, or stimuli into cell cycle exposure to ultraviolet light activity through a mulCAPSULE SUMMARY to develop melanoma.11 The tistep process. presence of H-RAS mutations d Mutations at several loMelanomas develop from familial in Spitz nevi also supports cations in the pathway mutations, or mutations acquired from the need for additional muhave been found in melinsults to melanocytic DNA. tations to develop melaanoma, the most comBRAFv600E, NRAS, phosphatase with noma. H-RAS mutations are v600E . mon being BRAF tensin homolog, and KIT are all involved seen in 29% of Spitz nevi, in cell cycle activating pathways and are 14% of atypical Spitz nevi, The MAPK pathway concommonly mutated in melanomas. More and 7% of Spitzoid lesions tains the proteins RAS, RAF, than one mutation is likely needed to worrisome for melanoma. In MEK, and ERK and is part of a transform melanocytes into melanoma. all, 86% of Spitzoid melanosignaling cascade that uses a mas have B-RAF or N-RAS chain of phosphorylation to Mutation-specific treatments such as mutations.4 This mutation transfer extracellular signals dual-drug therapy with tyrosine kinase profile may prove helpful in from the cell membrane to inhibitors of two components of the differentiating an atypical the nucleus6-8 (Fig 1). The mitogen-activated protein kinase Spitz nevus from a Spitzoid successful activation of the pathway and imatinib can be very melanoma.4 pathway activates genes in effective. The RAF proteins consist the nucleus that promote of A-RAF, B-RAF, and C-RAF, cell proliferation.3 Although which are protein kinases.12 B-RAF mutations are RAS and RAF mutations have been well documented 6,7,9 found in 66% of melanomas, and 90% of those mutations at each level can theoin melanoma, B-RAF mutations are a T to A substitution at codon retically activate the pathway and play a role in 6003,8 (Fig 2). This mutation promotes tumor oncogenesis. This pathway is complicated by the growth through cell proliferation and increased multiple isoforms of the RAS, RAF, MEK, and ERK angiogenesis via increased vascular endothelial proteins that each have functions and many regulagrowth factor and macrophage inhibitory tory aspects that are yet unknown. cytokine-1. B-RAFv600E also regulates interleukin-8, The RAS family of proteins contains N-RAS, 3,7 which promotes melanocyte adhesion to the vasK-RAS, and H-RAS. The RAS family has several culature, helping to promote metastases.3 downstream targets including RAF and PI3K.7 In contrast to this, B-RAF mutations are also seen Stimulation of either pathways lead to activation of in typical and atypical melanocytic nevi.8 In nevi, cyclin D (CCND1), which couples with cyclinB-RAF mutations initially trigger growth in lesions dependent kinase (CDK)-4 to stimulate cell proliferthat will eventually stop proliferating and remain ation.8,10 Mutations in each of the 3 RAS proteins has benign.13 The term ‘‘senescence pathway’’ describes been documented in melanoma.11 this benign behavior, which is theorized to occur in N-RAS mutations have been found in 15% to 22% the 82% of melanocytic nevi that have B-RAF mutaof melanomas6,8,11 and at one time were thought to tions.14 In light of this, a second mutation causing the be the most common mutations in melanoma.4 Most loss of a tumor suppressor gene or the gain of a mutations in N-RAS occur at codon 61 and render second mutation could cause a transition from nevi the protein active.6 Mouse models have shown a with mutated B-RAF to melanoma.3,8,13,15 This proneed for concurrent loss of p16, a tumor suppressor 11 gression has been referred to as the proliferation gene, to develop melanoma. Studies have also pathway.14 The second stimuli that is needed to shown us that N-RAS-mutated cells are more tu11 progress from benign nevi to melanoma is not fully morigenic than K-RAS cells. N-RAS mutations are understood at this time.13 usually not seen in cells with B-RAF mutations and Mutated B-RAF is just one pathway to melanoma. are accepted to be mutually exclusive with very rare Researchers have described a divergent pathway to exceptions.6 d
d
d
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Fig 1. Mitogen-activated protein kinase and phosphatidylinositol 3’-kinase pathways (PI3K ). CDK, Cyclindependent kinase; MITF, microphthalmia-associated transcriptional factor; PTEN, phosphatase with tensin homolog.
melanoma that starts with sun exposure to normal cells and diverges based on what mutations those cells accumulate. The hope is that we will be able to recognize clinical and microscopic patterns for the major melanoma mutations. An example of this is evidence that B-RAF mutations often occur in people with high levels of ultraviolet exposure in early life compared to patients with N-RAS mutations who have high total exposure spread throughout life. The B-RAF-mutated lesions also have specific characteristics microscopically such as larger, rounder, and more pigmented melanocytes; increased pagetoid spread; acanthosis; and sharper demarcation.16,17 Clinically, patients with B-RAF mutations are younger and have greater numbers of nevi. Patients with long-term total lifetime sun exposure were less likely to have B-RAF mutations, an example of this being a low percentage of lentigo maligna with B-RAF mutations.16 There is much left to learn about the B-RAFv600E mutation. Although it is present in many melanomas, the mutation alone seems to only cause benign proliferation and then senescence. Further studies will be needed to determine what specifically begins the change from senescence to malignancy. A-RAF and C-RAF mutations are not considered to be common causes of melanoma; however, one study has shown that B-RAF-inhibited cells with RAS mutations can use C-RAF to continue the proliferation signal through the MAPK pathway.12 This one example demonstrates how versatile the pathways are and how much we have to learn about their regulation and adaptability. MEK-1 and -2 are protein kinases that are downstream of B-RAF. MEK is active in 30% of all cancers. Inhibitors for the protein are being developed as therapeutic targets for B-RAF-mutated melanomas.3 ERK-1 and -2 are the only proteins downstream of MEK and are upstream of microphthalmia-associated transcriptional factor, which is a regulator of melanocyte differentiation.8 ERK activation can be seen in melanoma whereas lower levels of activated ERK are seen in melanocytes in normal-appearing skin. One study showed ERK activity from 40% to 100% in all of
Fig 2. Depiction of location of BRAF gene on chromosome 7. Image from Genetics Home Reference. Bethesda: US National Library of Medicine. 2003. Available from: URL:http://ghr.nlm.nih.gov/gene/BRAF. Accessed November 11, 2010.
the melanomas examined. Because ERK activation is seen in benign and malignant melanocytes, it is likely a marker of disease progression, but not a cause of melanoma.3
PI3K AND PHOSPHATASE WITH TENSIN HOMOLOG Key points d
d
PI3K begins a second pathway that can be activated by RAS. Phosphatase with tensin homolog (PTEN) mutations are seen in 40% of melanomas.
PI3K is a downstream effector of RAS and triggers a second pathway that also ends in cyclin-D activation and cell proliferation, similar to the MAPK pathway. PI3K mutations in melanoma are rare; however, disruption of the pathway is common through mutations in PTEN. PTEN is a downregulator of AKT (Fig 1). Mutations of PTEN are seen in 40% of melanomas7 and these melanomas are often found to have increased levels of invasion.8
P53 AND P16INK4A/RETINOBLASTOMA TUMOR SUPPRESSOR PATHWAYS Key points d
Mutations in CDKN2A are inherited and found in 40% of families with multiple cases of melanoma.
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Fig 4. P53 and p16INK4a/retinoblastoma ( pRB) pathways. CDK, Cyclin-dependent kinase.
Fig 3. Depiction of location of cyclin-dependent kinase (CDK )-N2A gene on chromosome 9. Image from Genetics Home Reference. Bethesda: US National Library of Medicine. 2003. Available from: URL:http://ghr.nlm.nih.gov/ gene/CDKN2A. Accessed November 11, 2010.
without the presence of p16.13,15 Much is left to learn. As more targeted pharmacotherapy is developed, understanding this pathway will be vital to appropriate treatment of patients in multiple-case melanoma families.
TREATMENT POSSIBILITIES Key points d
d d
P16, p53, p16INK4a/retinoblastoma, and p14 are tumor suppressors that likely interact with BRAF mutations in the senescence and proliferation pathways.
Tumor suppressor pathways play a large role in inherited and somatically acquired melanoma mutations. CDKN2A mutations are found in 40% of families with multiple cases of melanoma (Fig 3). CDKN2A encodes tumor suppressors p16INK4a and p14ARF. The majority of mutations in CDKN2A affect p16 whereas only 2% of at-risk melanoma families have an ARF mutation.18 Although much is known about this pathway, all of the cross-interactions are not understood. P16 functions to inhibit CDK-4 and keep the retinoblastoma protein in its suppressive state in which it functions to halt the cell cycle18 (Fig 4). These actions help maintain N-RAS senescence and seem to play a role in B-RAF senescence as well.3,8,15 P53 is a tumor suppressor activated by DNA damage and cellular stress.19 When activated it has many downstream effects including induction of p21, which helps maintain the suppressor activity of p16INK4a/retinoblastoma.15 Although this pathway seems to be linear and straightforward, it is not. Downstream proteins are still effective when p53 is inactivated, and B-RAF-mutated nevi can still achieve senescence
d
Many new melanoma treatments inhibit one member of the MAPK pathway. The most successful developmental treatment methods will likely use two-drug therapy to inhibit two steps in the MAPK pathway. Imatinib is effective against melanomas with KIT mutations. As we detect more common mutations, we may find that drugs we already have are also effective treatments.
As we learn more about melanoma mutations we will be able to develop new mutation-specific medications and take advantage of existing medications. Many of the new drugs are inhibitors of the MAPK pathway (Fig 5). PI3K, MEK, and RAF kinase inhibitors are currently being developed or tested individually and in combination. These drugs have had successes and setbacks. Individually they are plagued with the development of tumor resistance and unknown long-term side effects. An example is a BRAF inhibitor, PLX4032, which has activity against BRAFv600E. In early tests, the drug had 48% of B-RAF mutation-positive patients experience 50% or greater regression of their disease. Fifty eight percent of B-RAF mutation-positive patients had 30% or greater regression. In its phase I trial, patients who responded had progression-free survival of 8 to 9 months.20 There is a theoretical risk that the drug could increase the cell cycle in melanoma cells that have an N-RAS mutation. Up-regulation of NRAS,
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superior diagnosis and care for patients with melanoma.
Fig 5. Mitogen-activated protein kinase pathway and related medications. CDK, Cyclin-dependent kinase; MITF, microphthalmia-associated transcriptional factor; PI3K, phosphatidylinositol 3’-kinase; PTEN, phosphatase with tensin homolog.
and therefore activation of the MAPK pathway, by PLX4032 is another method for resistance.21 These issues are being addressed with multiple-drug therapy. Examples of this are trials adding a MEK and RAF inhibitor, trial NCT01072175, and planned trials that would combine RAF inhibitors with PI3K or other MEK inhibitors.20 Some known medications have been used for melanoma after finding susceptible mutation patterns. After identifying KIT mutations in mucosal (21%), acral (11%), and melanomas on chronic sun-damaged skin (17%) using molecular genetics techniques, researchers recognized the potential for an effective treatment using imatinib. Imatinib is an inhibitor of several tyrosine kinases including KIT. Of patients with gastrointestinal stromal tumors, 80% have improved disease after taking imatinib. Early results of treatment with imatinib for metastatic mucosal and acral lentiginous melanomas in patients with KIT gene mutations in either exons 11 or 13 showed a near complete response to treatment.5 A larger trial had 83% of its patients with stable disease to complete remission.22 Compared with the older treatments used today these numbers are very encouraging. As we begin to understand each mutation and pathway better, we look toward being able to use molecular analysis to make accurate histopathologic diagnosis based on mutations present, and to translating those mutations into tumor-specific treatment regimens.12 With the evidence of multiple mutations driving a single tumor, whether from clonality or multiple mutations in one cell line, it is clear that the best treatments will be combinations of several medications.12 Although several inhibitors are being tested with promising initial data, there is still much to learn. One important question is what other effects these medications will have on malignant and benign cells. Will inhibitors of malignant cells somehow cause replication of previously benign cells?12 Only time and further research will reveal these answers and show how these vast gains in technology will translate into
REFERENCES 1. National Cancer Institute. Melanoma home page. Available from URL: http://www.cancer.gov/cancertopics/types/melanoma. Accessed May 16, 2010. 2. Shoo A, Sagebiel R, Kashani-Sabet M. Discordance in the histopathologic diagnosis of melanoma at a melanoma referral center. J Am Acad Dermatol 2010;62:751-6. 3. Inamdar G, Madhunapantula S, Robertson G. Targeting the MAPK pathway in melanoma: why some approaches succeed and other fail. Biochem Pharmacol 2010;80:624-37. 4. van Dijk M, Bernsen M, Ruiter D. Analysis of mutations in B-RAF, N-RAS, and H-RAS genes in the differential diagnosis of Spitz nevus and Spitzoid melanoma. Am J Surg Pathol 2005; 29:1145-51. 5. Garrido M, Bastian B. KIT as a therapeutic target in melanoma. J Invest Dermatol 2010;130:20-7. 6. Jovanovic B, Egyhazi S, Eskandarpour M, Ghiorzo P, Palmer JM, Bianchi Scarra G, et al. Coexisting NRAS and BRAF mutations in primary familial melanomas with specific CDKN2A germline alterations. J Invest Dermatol 2010;130: 618-20. 7. Mishra PJ, Ha L, Rieker J, Sviderskaya EV, Bennett DC, Oberst MD, et al. Dissection of RAS downstream pathways in melanomagenesis: a role for Ral in transformation. Oncogene 2010;29:2449-56. 8. Takata M, Saida T. Genetic alterations in melanocytic tumors. J Dermatol Sci 2006;43:1-10. 9. Dong J, Phelps R, Qiao R, Yao S, Benard O, Ronai Z, et al. BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma. Cancer Res 2003;63: 3883-5. 10. Zhong Z, Yeow WS, Zou C, Wassell R, Wang C, Pestell RG, et al. Cyclin D1/cyclin-dependent kinase 4 interacts with filamin A and affects the migration and invasion potential of breast cancer cells. Cancer Res 2010;70:2105-14. 11. Milagre C, Dhomen N, Geyer F, Hayward R, Lambros M, Reis-Filho JS, et al. A mouse model of melanoma driven by oncogenic KRAS. Cancer Res 2010;70:5549-57. 12. Heidorn S, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas L, Dhomen N, et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 2010;140:209-21. 13. Michaloglou C, Vredeveld L, Soengas M, Denoyelle C, Kuilman T, van der Horst CM, et al. BRAFE600-associated senescencelike cell cycle arrest of human nevi. Nature 2005;436: 720-4. 14. Wajapeyee N, Serra R, Zhu X, Mahalingam M, Green MR. Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 2008; 132:363-74. 15. Haferkamp S, Tran S, Becker T, Scurr L, Kefford RF, Rizos H. The relative contributions of the p53 and pRb pathways in oncogene induced melanocyte senescence. Aging (Albany NY) 2009;1:542-56. 16. Hacker E, Hayward N, Dumenil T, James M. The association between MC1R genotype and BRAF mutation status in cutaneous melanoma: findings from an Australian population. J Invest Dermatol 2010;130:241-8. 17. Viros A, Fridlyand J, Bauer J, Lasithiotakis K, Garbe C, Pinkel D, Bastian BC. Improving melanoma classification by integrating genetic and morphologic features. PLoS Med 2008;6: e120.
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18. McKenzie H, Fung C, Becker T, Irvine M, Mann G, Kefford R, et al. Predicting functional significance of cancer-associated p16INK4a mutations in CDKN2A. Hum Mutat 2010;31:692-701. 19. Box N, Terzian T. The role of p53 in pigmentation, tanning and melanoma. Pigment Cell Melanoma Res 2008;21:525-33. 20. Arkenau HT, Kefford R, Long GV. Targeting BRAF for patients with melanoma. Br J Cancer 2011;104:392-8.
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21. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H, et al. Melanomas acquire resistance to B-RAF (V600E) inhibition by RTK of N-RAS up-regulation. Nature 2010; 468:973-7. 22. Woodman S, Davies M. Targeting KIT in melanoma: a paradigm of molecular medicine and targeted therapeutics. Biochem Pharmacol 2010;80:568-74.
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n 2010 there were an estimated 68,130 new cases of melanoma and 8700 deaths from melanoma in the United States.1 At this point in time early detection and excision is our best hope for prolonged survival. The difficulties of melanoma do not stop with awareness and detection but extend to the diagnosis of histologically ambiguous lesions. Accurate microscopic diagnosis of atypical melanocytic lesions is very challenging and discordance in diagnoses has been shown between dermatopathologists, who are experts in the diagnosis of melanocytic neoplasms.2 Great strides have been made in understanding the molecular pathways and mutations from which many melanomas originate. The hope of this research is to identify specific mutations that may be useful as genetic markers to aid in histopathologic diagnosis and targeted mutation-specific treatments.3,4 To understand how individual mutations can be used for diagnosis and treatment we must first review the most commonly mutated genes and genetic pathways.
KIT Key point d
KIT mutations are found in acral and mucosal melanomas, and in melanomas on chronically sun-damaged skin.
From the Department of Dermatology, Medical University of South Carolina,a and DermPath Diagnostics of South Carolina.b Funding sources: None. Conflicts of interest: None declared. Accepted for publication June 7, 2011.
Abbreviations used: CDK: MAPK: PI3K: PTEN:
cyclin-dependent kinase mitogen-activated protein kinase phosphatidylinositol 3’-kinase phosphatase with tensin homolog
The KIT gene encodes a tyrosine kinase membrane receptor that stimulates the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3’-kinase (PI3K) pathways (Fig 1). This gene has long been known to be involved in melanocyte development because of the lack of migration and disappearance of KIT-deficient melanoblasts, as well as the relationship of KIT with melanocytes seen in piebaldism. Studies have shown that KIT activation stimulates melanocyte migration more than proliferation.5 While studying acral melanomas, researchers have found melanocytes with similar chromosomal mutations, but appropriate numbers and spacing, up to 2 cm beyond the melanoma itself.5 Studies have also found a common location for mutation in acral melanomas using molecular genetics in chromosome 4q. This location includes the KIT gene.5 Researchers then began to look for KIT mutations in melanoma using molecular technology techniques and found them present in 11% of acral, 21% of mucosal, and 17% of melanomas appearing
Reprint requests: Julie M. Swick, MD, Department of Dermatology, Medical University of South Carolina, 135 Rutledge Ave, 11th Floor, Charleston, SC 29425. E-mail: [email protected]. Published online March 29, 2012. 0190-9622/$36.00 Ó 2012 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2011.06.047
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on chronically sun-damaged sites. This was in comparison with nonchronic sun-damaged melanomas that often had B-RAF mutations.5
K-RAS mutations have been found in 2% of melanomas. Although this protein plays a very active role in other malignancies, such as pancreatic and colon cancers, the same has not been true for melanoma.11 MAPK PATHWAY H-RAS mutations are present in 1% of melanomas. Key points Mouse models have shown that H-RAS-mutated mice d The MAPK pathway transfers extracellular require a loss of p16, p53, or stimuli into cell cycle exposure to ultraviolet light activity through a mulCAPSULE SUMMARY to develop melanoma.11 The tistep process. presence of H-RAS mutations d Mutations at several loMelanomas develop from familial in Spitz nevi also supports cations in the pathway mutations, or mutations acquired from the need for additional muhave been found in melinsults to melanocytic DNA. tations to develop melaanoma, the most comBRAFv600E, NRAS, phosphatase with noma. H-RAS mutations are v600E . mon being BRAF tensin homolog, and KIT are all involved seen in 29% of Spitz nevi, in cell cycle activating pathways and are 14% of atypical Spitz nevi, The MAPK pathway concommonly mutated in melanomas. More and 7% of Spitzoid lesions tains the proteins RAS, RAF, than one mutation is likely needed to worrisome for melanoma. In MEK, and ERK and is part of a transform melanocytes into melanoma. all, 86% of Spitzoid melanosignaling cascade that uses a mas have B-RAF or N-RAS chain of phosphorylation to Mutation-specific treatments such as mutations.4 This mutation transfer extracellular signals dual-drug therapy with tyrosine kinase profile may prove helpful in from the cell membrane to inhibitors of two components of the differentiating an atypical the nucleus6-8 (Fig 1). The mitogen-activated protein kinase Spitz nevus from a Spitzoid successful activation of the pathway and imatinib can be very melanoma.4 pathway activates genes in effective. The RAF proteins consist the nucleus that promote of A-RAF, B-RAF, and C-RAF, cell proliferation.3 Although which are protein kinases.12 B-RAF mutations are RAS and RAF mutations have been well documented 6,7,9 found in 66% of melanomas, and 90% of those mutations at each level can theoin melanoma, B-RAF mutations are a T to A substitution at codon retically activate the pathway and play a role in 6003,8 (Fig 2). This mutation promotes tumor oncogenesis. This pathway is complicated by the growth through cell proliferation and increased multiple isoforms of the RAS, RAF, MEK, and ERK angiogenesis via increased vascular endothelial proteins that each have functions and many regulagrowth factor and macrophage inhibitory tory aspects that are yet unknown. cytokine-1. B-RAFv600E also regulates interleukin-8, The RAS family of proteins contains N-RAS, 3,7 which promotes melanocyte adhesion to the vasK-RAS, and H-RAS. The RAS family has several culature, helping to promote metastases.3 downstream targets including RAF and PI3K.7 In contrast to this, B-RAF mutations are also seen Stimulation of either pathways lead to activation of in typical and atypical melanocytic nevi.8 In nevi, cyclin D (CCND1), which couples with cyclinB-RAF mutations initially trigger growth in lesions dependent kinase (CDK)-4 to stimulate cell proliferthat will eventually stop proliferating and remain ation.8,10 Mutations in each of the 3 RAS proteins has benign.13 The term ‘‘senescence pathway’’ describes been documented in melanoma.11 this benign behavior, which is theorized to occur in N-RAS mutations have been found in 15% to 22% the 82% of melanocytic nevi that have B-RAF mutaof melanomas6,8,11 and at one time were thought to tions.14 In light of this, a second mutation causing the be the most common mutations in melanoma.4 Most loss of a tumor suppressor gene or the gain of a mutations in N-RAS occur at codon 61 and render second mutation could cause a transition from nevi the protein active.6 Mouse models have shown a with mutated B-RAF to melanoma.3,8,13,15 This proneed for concurrent loss of p16, a tumor suppressor 11 gression has been referred to as the proliferation gene, to develop melanoma. Studies have also pathway.14 The second stimuli that is needed to shown us that N-RAS-mutated cells are more tu11 progress from benign nevi to melanoma is not fully morigenic than K-RAS cells. N-RAS mutations are understood at this time.13 usually not seen in cells with B-RAF mutations and Mutated B-RAF is just one pathway to melanoma. are accepted to be mutually exclusive with very rare Researchers have described a divergent pathway to exceptions.6 d
d
d
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Fig 1. Mitogen-activated protein kinase and phosphatidylinositol 3’-kinase pathways (PI3K ). CDK, Cyclindependent kinase; MITF, microphthalmia-associated transcriptional factor; PTEN, phosphatase with tensin homolog.
melanoma that starts with sun exposure to normal cells and diverges based on what mutations those cells accumulate. The hope is that we will be able to recognize clinical and microscopic patterns for the major melanoma mutations. An example of this is evidence that B-RAF mutations often occur in people with high levels of ultraviolet exposure in early life compared to patients with N-RAS mutations who have high total exposure spread throughout life. The B-RAF-mutated lesions also have specific characteristics microscopically such as larger, rounder, and more pigmented melanocytes; increased pagetoid spread; acanthosis; and sharper demarcation.16,17 Clinically, patients with B-RAF mutations are younger and have greater numbers of nevi. Patients with long-term total lifetime sun exposure were less likely to have B-RAF mutations, an example of this being a low percentage of lentigo maligna with B-RAF mutations.16 There is much left to learn about the B-RAFv600E mutation. Although it is present in many melanomas, the mutation alone seems to only cause benign proliferation and then senescence. Further studies will be needed to determine what specifically begins the change from senescence to malignancy. A-RAF and C-RAF mutations are not considered to be common causes of melanoma; however, one study has shown that B-RAF-inhibited cells with RAS mutations can use C-RAF to continue the proliferation signal through the MAPK pathway.12 This one example demonstrates how versatile the pathways are and how much we have to learn about their regulation and adaptability. MEK-1 and -2 are protein kinases that are downstream of B-RAF. MEK is active in 30% of all cancers. Inhibitors for the protein are being developed as therapeutic targets for B-RAF-mutated melanomas.3 ERK-1 and -2 are the only proteins downstream of MEK and are upstream of microphthalmia-associated transcriptional factor, which is a regulator of melanocyte differentiation.8 ERK activation can be seen in melanoma whereas lower levels of activated ERK are seen in melanocytes in normal-appearing skin. One study showed ERK activity from 40% to 100% in all of
Fig 2. Depiction of location of BRAF gene on chromosome 7. Image from Genetics Home Reference. Bethesda: US National Library of Medicine. 2003. Available from: URL:http://ghr.nlm.nih.gov/gene/BRAF. Accessed November 11, 2010.
the melanomas examined. Because ERK activation is seen in benign and malignant melanocytes, it is likely a marker of disease progression, but not a cause of melanoma.3
PI3K AND PHOSPHATASE WITH TENSIN HOMOLOG Key points d
d
PI3K begins a second pathway that can be activated by RAS. Phosphatase with tensin homolog (PTEN) mutations are seen in 40% of melanomas.
PI3K is a downstream effector of RAS and triggers a second pathway that also ends in cyclin-D activation and cell proliferation, similar to the MAPK pathway. PI3K mutations in melanoma are rare; however, disruption of the pathway is common through mutations in PTEN. PTEN is a downregulator of AKT (Fig 1). Mutations of PTEN are seen in 40% of melanomas7 and these melanomas are often found to have increased levels of invasion.8
P53 AND P16INK4A/RETINOBLASTOMA TUMOR SUPPRESSOR PATHWAYS Key points d
Mutations in CDKN2A are inherited and found in 40% of families with multiple cases of melanoma.
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Fig 4. P53 and p16INK4a/retinoblastoma ( pRB) pathways. CDK, Cyclin-dependent kinase.
Fig 3. Depiction of location of cyclin-dependent kinase (CDK )-N2A gene on chromosome 9. Image from Genetics Home Reference. Bethesda: US National Library of Medicine. 2003. Available from: URL:http://ghr.nlm.nih.gov/ gene/CDKN2A. Accessed November 11, 2010.
without the presence of p16.13,15 Much is left to learn. As more targeted pharmacotherapy is developed, understanding this pathway will be vital to appropriate treatment of patients in multiple-case melanoma families.
TREATMENT POSSIBILITIES Key points d
d d
P16, p53, p16INK4a/retinoblastoma, and p14 are tumor suppressors that likely interact with BRAF mutations in the senescence and proliferation pathways.
Tumor suppressor pathways play a large role in inherited and somatically acquired melanoma mutations. CDKN2A mutations are found in 40% of families with multiple cases of melanoma (Fig 3). CDKN2A encodes tumor suppressors p16INK4a and p14ARF. The majority of mutations in CDKN2A affect p16 whereas only 2% of at-risk melanoma families have an ARF mutation.18 Although much is known about this pathway, all of the cross-interactions are not understood. P16 functions to inhibit CDK-4 and keep the retinoblastoma protein in its suppressive state in which it functions to halt the cell cycle18 (Fig 4). These actions help maintain N-RAS senescence and seem to play a role in B-RAF senescence as well.3,8,15 P53 is a tumor suppressor activated by DNA damage and cellular stress.19 When activated it has many downstream effects including induction of p21, which helps maintain the suppressor activity of p16INK4a/retinoblastoma.15 Although this pathway seems to be linear and straightforward, it is not. Downstream proteins are still effective when p53 is inactivated, and B-RAF-mutated nevi can still achieve senescence
d
Many new melanoma treatments inhibit one member of the MAPK pathway. The most successful developmental treatment methods will likely use two-drug therapy to inhibit two steps in the MAPK pathway. Imatinib is effective against melanomas with KIT mutations. As we detect more common mutations, we may find that drugs we already have are also effective treatments.
As we learn more about melanoma mutations we will be able to develop new mutation-specific medications and take advantage of existing medications. Many of the new drugs are inhibitors of the MAPK pathway (Fig 5). PI3K, MEK, and RAF kinase inhibitors are currently being developed or tested individually and in combination. These drugs have had successes and setbacks. Individually they are plagued with the development of tumor resistance and unknown long-term side effects. An example is a BRAF inhibitor, PLX4032, which has activity against BRAFv600E. In early tests, the drug had 48% of B-RAF mutation-positive patients experience 50% or greater regression of their disease. Fifty eight percent of B-RAF mutation-positive patients had 30% or greater regression. In its phase I trial, patients who responded had progression-free survival of 8 to 9 months.20 There is a theoretical risk that the drug could increase the cell cycle in melanoma cells that have an N-RAS mutation. Up-regulation of NRAS,
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superior diagnosis and care for patients with melanoma.
Fig 5. Mitogen-activated protein kinase pathway and related medications. CDK, Cyclin-dependent kinase; MITF, microphthalmia-associated transcriptional factor; PI3K, phosphatidylinositol 3’-kinase; PTEN, phosphatase with tensin homolog.
and therefore activation of the MAPK pathway, by PLX4032 is another method for resistance.21 These issues are being addressed with multiple-drug therapy. Examples of this are trials adding a MEK and RAF inhibitor, trial NCT01072175, and planned trials that would combine RAF inhibitors with PI3K or other MEK inhibitors.20 Some known medications have been used for melanoma after finding susceptible mutation patterns. After identifying KIT mutations in mucosal (21%), acral (11%), and melanomas on chronic sun-damaged skin (17%) using molecular genetics techniques, researchers recognized the potential for an effective treatment using imatinib. Imatinib is an inhibitor of several tyrosine kinases including KIT. Of patients with gastrointestinal stromal tumors, 80% have improved disease after taking imatinib. Early results of treatment with imatinib for metastatic mucosal and acral lentiginous melanomas in patients with KIT gene mutations in either exons 11 or 13 showed a near complete response to treatment.5 A larger trial had 83% of its patients with stable disease to complete remission.22 Compared with the older treatments used today these numbers are very encouraging. As we begin to understand each mutation and pathway better, we look toward being able to use molecular analysis to make accurate histopathologic diagnosis based on mutations present, and to translating those mutations into tumor-specific treatment regimens.12 With the evidence of multiple mutations driving a single tumor, whether from clonality or multiple mutations in one cell line, it is clear that the best treatments will be combinations of several medications.12 Although several inhibitors are being tested with promising initial data, there is still much to learn. One important question is what other effects these medications will have on malignant and benign cells. Will inhibitors of malignant cells somehow cause replication of previously benign cells?12 Only time and further research will reveal these answers and show how these vast gains in technology will translate into
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