Layfield Wallander USCAP KIT mutations by fxz3722


									                                                                                    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
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).
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.
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.
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
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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