KIT TARGETED MUTATION ANALYSIS FOR MALIGNANT MELANOMA
Label Name: KITMEL
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6185 
Last Review:  3/17/2017 10:04:00 AM
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years CHECK WITH LABORATORY 1  ML
Collection Notes
  Adult:
  • Formalin Fixed Paraffin Embedded Tissue: The laboratory can receive either a paraffin embedded tissue block or four freshly cut (within one week)5uM thick unstained slides containing 3 to 20 square mm of tissue. Unstained slides should be accompanied by an H&E stained slide for histologic evaluation.
 
Transport
  Formalin fixed paraffin embedded tissue blocks and slides can be sent to the lab at ambient temperature.


Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values
KITMEL
NO MUTATIONS DETECTED
Methodology
  This assay uses PCR amplification followed by Sanger DNA sequencing to detect activating mutations in exons 9, 11, 13, 17 and 18 of the KIT gene. An H&E stained slide for each case is first evaluated to identify the regions of greatest tumor content. These regions are then macro-dissected from adjacent unstained formalin-fixed paraffin-embedded sections and used to prepare genomic DNA. The protein coding and flanking intronic sequences of exons 9, 11, 13, 17 and 18 of the KIT gene are amplified from this purified genomic DNA by PCR. The primers used in these PCR reactions contain M13 universal primer “tails” at their 5’ ends, and have 3’ ends that are complementary to their genomic target sequence. The resulting PCR products are treated with an exonuclease/ phosphatase mixture (ExoSAP-IT) to remove excess PCR primers and nucleotides. These purified DNA amplicons are then sequenced using universal M13 forward and reverse sequencing primers (M13 Forward/-20 and M13 Reverse/-27) and the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The products of the completed sequencing reactions are purified with the Big Dye XTerminator Purification Kit and resolved using the ABI 3130xl Genetic Analyzer. Data is analyzed using the ABI Data Collection software v3.0, Sequencing Analysis software 5.4 and SeqScape software v2.6. Sequences are compared to the reference DNA sequence (GenBank Accession: NM_000222.2) for the KIT gene.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
   
   
Clinical Significance and Interpretive Data
    KIT is a tyrosine kinase receptor that regulates cellular proliferation and differentiation. Activating mutations in KIT have been identified in melanomas. Specifically, 17% of sun damage associated, 11-23% of acral, 8% of conjunctival and 15-21% of mucosal melanomas harbor KIT mutations. Mutations have been documented in both the juxtamembrane and kinase domains of KIT.

There are several case reports of melanoma patients with significant clinical responses to treatment with small molecule tyrosine kinase inhibitors including imatinib, sorafenib and dasatinib. Mutations in the KIT juxtamembrane domain (within exon 11) were identified in these patients’ tumors. In addition, a forty three patient, phase II, single arm trial in patients with metastatic melanoma harboring mutations in exons 11 and 13 of KIT demonstrated significant clinical response to imatinib. While these studies are preliminary, they suggest KIT mutation analysis may be helpful in determining the utility of targeted tyrosine kinase inhibitors in melanoma patients. However, multiple factors including clinical findings and the results of other laboratory tests contribute to utility, efficacy and appropriateness of tyrosine kinase inhibitors in treating melanoma patients. Thus, this test is not a substitute for a physician's judgment and clinical experience

References

Beadling C et al. KIT gene mutations and copy number in melanoma subtypes. Clin Cancer Res. 2008; 14: 6821–6828.

Curtin JA et al. Somatic activation of KIT in distinct subtypes of melanoma. J Clin Oncol 2006; 24: 4340–4346.

Fisher DE et al. Melanoma from bench to bedside: meeting report from the 6th international melanoma congress. Pigment Cell Melanoma Res 2010; 23: 14–26.

Guo J et el. Phase II, open-label, single-arm trial of imatinib mesylate in patients with metastatic melanoma harboring c-Kit mutation or amplification. J Clin Oncol. 2011; 29: 2904-2909.

Hodi FS et al. Major response to imatinib mesylate in KIT-mutated melanoma. J Clin Oncol 2008; 26: 2046–2051.

Quintas-Cardama A et al. Complete response of stage IV anal mucosal melanoma expressing KIT Val560Asp to the multikinase inhibitor sorafenib. Nat Clin Pract Oncol 2008; 5: 737–740.

Woodman SE et al. Activity of dasatinib against L576P KIT mutant melanoma: molecular, cellular, and clinical correlates. Mol Cancer Ther 2009; 8: 2079–2085.

   
Indications
    Patients with metastatic melanoma harboring mutations in exons 11 and 13 of KIT demonstrated significant clinical response to treatment with small molecule tyrosine kinase inhibitors.
   
Limitations
    The sensitivity and specificity of DNA sequencing is high for the detection of nucleotide base changes, small deletions, and insertions in the regions analyzed. However, this assay may not detect an acquired mutation which is present below the 15% detection limit (i.e., mutant cell population of < 30%). Only exons 9, 11, 13, 17 and 18 of KIT were examined. Changes outside of this region will not be detected. The presence of a mutant population containing a large deletion, duplication, insertion, or sequence alteration adversely affecting primer binding may not be identified using these methods. This test is subject to interference by various factors including PCR inhibitors, poor DNA quality and insufficient DNA quantity. These sources of interference usually produce an uninterpretable result rather and an inaccurate result. Although rare, mutations or polymorphisms occurring in the primer binding regions can affect testing and could produce an inaccurate result.
   
Test Synonyms
  Synonym(s): c-KIT
Synonym(s): CKIT
Synonym(s): imatinib
Synonym(s): KIT
Synonym(s): melanoma
Molecular Diagnostics Laboratory
(MDX)

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

Address: 
 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: