KIT TARGETED MUTATION ANALYSIS FOR GIST
Label Name: KITGIST
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6184 
Last Review:  3/17/2017 10:04:12 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
KITGIST
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 approximately 75-80% of gastrointestinal stromal tumors (GISTs). These mutations occur in the juxtamembrane domain (exon 11; 80% of gastric GIST, 50% of small intestinal GIST), the extracellular domain (exon 9; 3% of gastric GIST, 25% of small intestinal GIST) and the tyrosine kinase domain (exons 13 and 17). The presence of a KIT mutation in a spindle cell neoplasm suggests a diagnosis of GIST, but is not specific for this diagnosis. In addition, the absence of a KIT mutation does not exclude the diagnosis of GIST. Histologic and immunohistochemical correlation is required.

Many of the acquired mutations in KIT result in constitutive activation of its tyrosine kinase activity. This makes the use of targeted tyrosine kinase inhibitory agents, such as imatinib, an option for patients with tumors that harbor these mutations. Approximately eighty percent (75-90%) of GISTs respond to imatinib therapy. Patients with GISTs that harbor mutations within the juxtamembrane domain (exon 11) of KIT have, on average, better responses to imatinib than patients with GISTs that harbor mutations in the KIT extracellular domain (exon 9). Multiple factors including clinical findings and the results of other laboratory tests contribute to utility, efficacy and appropriateness of the use of tyrosine kinase inhibitors in treating GIST patients. Thus, this test is intended for use as an aid in making individualized patient treatment decisions and is not a substitute for a physician's judgment and clinical experience.

Clinical resistance to imatinib can occur with prolonged treatment with imatinib due to secondary kinase domain mutations. These mutations are different than those tested in this assay, with the following exceptions: Secondary mutations (mutations acquired while on imatinib therapy) have been found in Exon 13 (Val654Ala, Val654Glu). These represent 33% of reported acquired secondary resistance mutations. Secondary mutations have been reported in Exon 17 (Cys809, Asp816, Lys818, Asp820, Asn822, Tyr823). These represent 52% of acquired resistance mutations. These mutations are occasionally seen as primary mutations. Secondary mutations in exon 14, 15, 16 have been described as resistance mutations. These are NOT detected in this assay. The most common of these are Thr670Glu and Thr670Ile in exon 14 and represents 14% of acquired resistance mutations.

References

Fletcher, JA and Rubin, BP. (2007) KIT mutations in GIST. Current Opinion in Genetics & Development 17:3-7.

Heinrich, MC, Corless, CL, Blanke, CD, Demetri,GD, Joensuu, H Roberts, PJ, Eisenberg, BL, von Mehren, M, Fletcher, CD and K Sandau., (2006) Molecular correlates of imatinib resistance in gastrointestinal stromal tumors, Journal of Clinical Oncology 24: 4764–4774.

Heinrich, MC, Corless, CL, Demetri,GD, Blanke, CD, von Mehren, M, Joensuu, H, McGreevey, LS, Chen, CJ, Van den Abbeele, AD, and Druker BJ et al. (2003) Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor, Journal of Clinical Oncology 21: 4342–4349.

Lasota, J and Miettinen, M. (2008) Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal tumors. Histopathology 53:245-266.

Debiec-Rychter M. et al., KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal tumors. Eur. J. Cancer 2006; 42; 1093-1103.

   
Indications
    Acquired mutations in KIT result in constitutive activation of its tyrosine kinase activity. This makes the use of targeted tyrosine kinase inhibitory agents, such as imatinib, an option for patients with tumors that harbor these mutations.
   
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.

Multiple factors including clinical findings and the results of other laboratory tests contribute to utility, efficacy and appropriateness of the use of tyrosine kinase inhibitors in treating GIST patients. Thus, this test is intended for use as an aid in making individualized patient treatment decisions and is not a substitute for a physician's judgment and clinical experience.
   
Test Synonyms
  Synonym(s): C-KIT
Synonym(s): CKIT
Synonym(s): gastrointestinal stromal tumor
Synonym(s): GIST
Synonym(s): imatinib
Synonym(s): KIT
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: