KIT Mutations in Ocular Melanoma Lester J. Layfield 1, Lyska L. Emerson1, Michelle L. Wallander2, Sheryl Tripp2, Don Davis3 and Nick Mamalis 3 1Department of Pathology, University of Utah Health Sciences Center and ARUP Laboratories, Salt Lake City, UT 2ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT 3Department of Ophthalmology & Visual Sciences, University of Utah School of Medicine, Salt Lake City, UT Introduction Results Results The prevalence of KIT mutations varies between melanoma KIT Exon 11: Sample OM #30 Eight of seventy-five (10.7%) ocular melanomas contained P573L subtypes, as defined by the site of anatomical origin. Acral, 100 mutations in either the KIT or PDGFRA gene. mucosal, and chronic sun damaged skin melanomas have been There was no significant correlation between mutational status Relative Signal (%) 80 Ex 11 Fwd KIT Ref. shown to harbor KIT mutations while non-chronic sun damaged and anatomical site (p = 0.393). 60 skin melanomas generally do not since they are often characterized OM #30 Ex 11 Fwd by BRAF or NRAS mutations. The frequency of KIT activating 40 CD117 positivity was approximately equal (82%) in melanomas of mutations in specific melanoma subtypes is relatively low with OM #30 Ex 11 Rev the choroid, iris and ciliary body. 20 reports of 15% for acral, 19% for mucosal, and 17% for chronic sun KIT expression was least prevalent in conjunctival melanomas 0 KIT Ref. Ex 11 Rev damaged skin melanomas. Ocular melanoma, although a rare (20%) but this failed to reach statistical significance (p = 0.641). melanoma subtype, does account for the majority of all intraocular 80 81 82 83 84 85 86 malignancies. Arising from melanocytes of the choroid, iris, ciliary CD117 positivity was not predictive of KIT mutational status. Temperature body and conjunctiva, ocular melanomas have been reported to Only 6 of 58 (10.3%) CD117 positive tumors harbored KIT express KIT (CD117) at frequencies between 63-91%. KIT R804W mutations. KIT Exon 17: Sample OM #14 mutations, however, have only been reported in one conjunctival tumor. Given the low frequency of KIT mutations (<1%) in ocular 100 One CD117 negative tumor harbored a KIT mutation. Relative Signal (%) KIT Ref. Ex 17 Fwd melanoma, it is not surprisingly that recent studies have failed to 80 Six of the eight mutations occurred in KIT exon 11. demonstrate the clinical efficacy for imatinib mesylate therapy in 60 OM #14 Ex 17 Fwd unselected patients. One mutation each was present in KIT exon 17 and PDGFRA 40 OM #14 Ex 17 Rev intron 18. As there are only limited data on the prevalence of KIT mutations in 20 ocular melanoma, we reviewed a large series of 75 ocular KIT Ref. Ex 17 Rev Conclusions 0 melanomas including cases arising in the choroid, iris, ciliary body 80 81 82 83 84 85 The high frequency of KIT overexpression in ocular melanoma has and conjunctiva. The frequency of KIT activating mutations and KIT Temperature led to an interest in the use of imatinib therapy for this melanoma protein expression was determined for each anatomical site. In subtype. However, overexpression of KIT in ocular melanoma does addition, we also investigated the frequency of platelet derived HRMA and sequencing results from two representative KIT mutated ocular melanoma (OM) samples. HRMA: negative controls not appear to imply response to imatinib. The likely explanation is growth factor receptor A (PDGFRA) mutations in ocular melanoma, (red), positive controls (blue), OM #30 (black) and OM #14 (green). the near absence of KIT activating mutations, as reported to date. which has not been reported to date. Only one conjunctival melanoma has been reported to harbor a KIT mutation. Our relatively large study set of ocular melanomas demonstrates that KIT mutations do occur in multiple ocular Materials and Methods Results anatomical locations at a frequency of 9.3%. All but one of our KIT mutations were located in exon 11, which 75 formalin-fixed, paraffin-embedded (FFPE) ocular melanomas encodes the juxtamembrane domain of the receptor. Given that this % CD117 % with % Mutation + of % Mutation + of Exons Involved domain normally functions as a negative regulator of kinase activity, were selected from the University of Utah Department of Tumor Site Ophthalmology files. Cases were reviewed to confirm the original Positive Mutations Total CD117 + Total CD117 - (Number of Cases) mutations that alter the conformation of this domain are therefore diagnosis and to ensure that sufficient tumor was present for KIT exon 11 (3) oncogenic. With the exception of the W582X mutation, we predict analysis. Choroidal 81.1% (43/53) 9.4% (5/53) 9.3% (4/43) 10% (1/10) KIT exon 17 (1) that the remaining exon 11 mutations are likely to be sensitive to imatinib. Conversely, the exon 17 R804W mutation is likely imatinib Choroidal: n = 53 PDGFRA intron 18 (1) resistant, given its location within the activation loop of KIT. We Iris 83.3% (5/6) 33.3% (2/6) 40.0% (2/5) 0% (0/1) KIT exon 11 (2) also identified a PDGFRA intron 18 mutation that may effect splicing Ciliary body: n = 11 Ciliary Body 81.8% (9/11) 9.1% (1/11) 11.1% (1/9) 0% (0/2) KIT exon 11 (1) due its location at the exon/intron boundary. Additional in vitro Iris: n = 6 Conjunctiva 20.0% (1/5) 0% (0/5) 0% (0/1) 0% (0/4) N/A studies would be needed to determine the efficacy of imatinib Conjunctiva: n = 5 Total 77.3% (58/75) 10.7% (8/75) 12.1% (7/58) 5.9% (1/17) against all of these mutations. KIT IHC: rabbit polyclonal CD117 (Dako) The utility of CD117 staining as a screen for KIT mutational status in ocular melanoma is limited. The majority of our cases were CD117 FFPE tumor tissue was macrodissected and DNA was extracted positive and mutation negative, yielding a positive predictive value by overnight proteinase K digestion. Mutation Location Ocular melanoma Mutations deduced at Mutations identified at Mutation Type of only 12% for CD117 staining. Our data suggests that screening subtype protein level DNA level at DNA level of all ocular melanomas for KIT and PDGFRA mutations by Crude DNA extract was used directly in PCR to amplify KIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18. mutational analysis is warranted to identify the approximately 10% KIT exon 11 Choroidal p.Val555Ile + p.Ser590Asn c.1684G>A (+) c.1790G>A Missense/Missense of patients that could benefit from imatinib therapy. Amplification and high resolution melting analysis (HRMA) were Choroidal p.Val569Ile + p.Ser590Asn c.1726G>A (+) c.1790G>A Missense/Missense performed on the LightScanner32 (Idaho Technology, Salt Lake City, Iris p.Asp572Asn c.1735G>A Missense References UT). Iris p.Pro573Leu c.1739C>T Missense Beadling C, Jacobson-Dunlop E, Hodi FS, et al. KIT gene mutations and copy number in Choroidal p.Pro573Leu c.1739C>T Missense Following amplification, samples were immediately melted by melanoma subtypes. Clin Cancer Res 2008;14:6821-8. Ciliary body p.Trp582X c.1767G>A Nonsense heating from 65°C to 95°C at 0.3°C/sec. Normalized and Torres-Cabala CA, Wang WL, Trent J, et al. Correlation between KIT expression and KIT mutation in melanoma: A study of 173 cases with emphasis on the acral-lentiginous/mucosal temperature-shifted melting curves were compared to wildtype DNA, KIT exon 17 Choroidal p.Arg804Trp c.2431C>T Missense type. Mod Pathol 2009;22:1446-56. which was used as a negative control. Hofmann UB, Kauczok-Vetter CS, Houben R et al. Overexpression of the KIT/SCF in uveal . melanoma does not translate into clinical efficacy of imatinib mesylate. Clin Cancer Res The use of human tissue for this analysis was approved by the PDGFRA intron 18 Choroidal c.2701+6G>A Splice site 2009;15:324-9. University of Utah Institutional Review Board (#22487). Wallander ML, Willmore-Payne C, Layfield LJ. C-KIT and PDGFRA zygosity in gastrointestinal stromal tumors: Correlation with tumor site, tumor size, exon, and CD117 immunohistochemistry. Appl Immunohistochem Mol Morphol 2011;19:21-7.
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