KIT Molecular Testing

Prognostication in CBF AML

  • Aids in the diagnosis of mastocytosis in peripheral blood and bone marrow specimens
  • The D816V mutation is a diagnostic marker for systemic mastocytosis
  • The D816V mutation confers resistance to imatinib
  • Detects activating mutations in KIT and PDGFRA
  • Predicts response to targeted therapy
  • Detects activating mutations in KIT and PDGFRA
  • Predicts response to targeted therapy
Related Tests

Assesses for multiple gene mutations, including substitutions and smaller insertions and deletions that may have prognostic and/or therapeutic significance

  • Identifies prognostically important abnormalities in newly diagnosed AML
  • Aids in classification of AML including subtypes with recurrent genetic abnormalities
  • Monitors response to therapy with specific probes (CHR FISHI) or progression of disease with probe panel

Aids in diagnosis and classification of hematologic neoplasms presenting with prominent eosinophilia

Initial screening test when GIST or mastocytosis is suspected based on histology and location of tumor

  • Screening test in tumor that is morphologically and clinically suspicious for GIST when CD117 is negative
  • Performed as a stain and return (technical) service only
  • Detects activating BRAF mutations at codon 600
  • Predicts response to targeted therapy in melanomas and colorectal cancers
  • Assesses prognosis of certain thyroid cancers
  • Determines BRAF V600E mutation status in patients with solid tumors to select candidates for targeted therapy with kinase inhibitors (BRAF and/or MEK)
  • Monitors response to therapy and disease progression in patients carrying BRAF V600E mutation

Molecular testing for KIT mutations is relevant for various types of cancer and can provide diagnostic, prognostic, and predictive information for systemic mastocytosis (SM), gastrointestinal stromal tumors (GIST), melanoma, and acute myeloid leukemia (AML) associated with inv(16) or t(8;21), also known as core-binding factor (CBF) AML. For GIST and melanoma, PDGFRA testing is also often relevant.

Disease Overview

Core-Binding Factor Acute Myeloid Leukemia

  • KIT mutation testing is important for prognostication 
  • KIT mutations are associated with higher incidence of relapse and lower survival 
  • KIT mutations may be detected in :
    • inv(16) or t(16;16) AML
    • t(8;21) AML

Mastocytosis

KIT mutation testing is important for:

  • Diagnosis (presence of mutation is a minor criteria for SM)
  • Prediction of response to targeted therapy

Gastrointestinal Stromal Tumors

  • KIT and PDGFRA mutation testing is important for prediction of response to targeted therapy and should be performed in all patients considered for targeted therapy
  • Mutation presence and type determine if the patient will benefit from targeted therapy
  • Detection of secondary resistance mutations in patient already treated with targeted therapy may guide the use of other therapeutic agents
  • Mutation testing may be occasionally used to aid in establishing GIST diagnosis in difficult cases (unusual location, morphology, or immunoprofile)
  • Immunohistochemistry for c-kit (CD117) is useful for diagnostic purposes but should not be used to predict response to targeted therapy

Melanoma

  • KIT mutation testing is important for prediction of response to targeted therapy
  • Immunohistochemistry for c-kit (CD117) should not be used to predict response to targeted therapy

Genetics

Gene

KIT

Structure/Function

  • Maps to 4q12
  • Receptor tyrosine kinase (type III)
  • Important in hematopoiesis for regulation of cell proliferation and maturation

Mutations

A variety of >500 mutations have been described, most commonly in juxtamembrane region (exon 11), extracellular region (exons 8, 9), and kinase domain (exons 13, 17). These mutations are commonly detected in patients with:

  • CBF AML
    • Detected in ~20% of AML with KIT mutations, including inv(16)
    • Detected in 20-25% of AML with t(8;21) (particularly the D816V mutation)
  • Mastocytosis
    • Adults
      • D816V mutation detected in 95% of patients with SM
      • Rare juxtamembrane mutations
    • Children
      • D816V mutation detected in 30-40% of patients with SM
      • ~40% carry KIT mutations that reside outside exon 17 (mainly exons 8 and 9)
    • Mutations other than D816V may be detected in SM-associated hematologic neoplasm (AHN)
  • GIST
    • KIT mutations present in ~85% of cases
      • Primary mutations most common in exon 11 (~70% of cases) and exon 9 (~10-15% of cases); much less common in other exons
      • Secondary resistance mutations occur in exons 13, 14, 17, and 18
    • PDGFRA mutations present in ~5% of cases
      • Primary mutations most common in exon 18 (~5% of cases)
      • Primary mutations much less common in other exons
  • Melanoma
    • KIT mutations present in 2-8% of cases overall (more common in mucosal and acral melanomas)
    • Most common in:
      • Exon 11 (70% of KIT mutated cases)
      • Exon 13 (20% of KIT mutated cases)
    • Much less common in other exons

Test Interpretation

KIT Mutations in AML by Fragment Analysis and Sequencing

Analytical Sensitivity

Detects mutations in:

  • Exon 17 in specimens with at least 30% AML cells harboring the mutation
  • Exon 8 in specimens with at least 5% AML cells harboring the mutation

Results

  • Detected: KIT exon 8 or 17 mutation
    • Associated with less favorable outcome in in CBF AML
    • TKIs may be useful in conjunction with standard chemotherapy
  • Not detected: no detectable mutation in KIT exon 8 or 17

Limitations

  • Not intended to detect minimal residual disease
  • Mutations outside of exons 8 and 17 are not detected
  • Mutations below analytical sensitivity will not be detected

KIT (D816V) Mutation by ddPCR, Quantitative

Sensitivity

  • Clinical: occurs in >80% of SM cases
  • Analytical: 0.03% variant allele frequency (VAF)

Results

  • Detected VAF: KIT (D816V) point mutation; allele specific amplification of the c.2447 C>T (D816V)
  • Results are reported as a percent mutated allele
    • Supports a diagnosis of SM or SM-associated clonal hematologic nonmast cell lineage disease (SM-AHNMD) in the correct clinical context
    • Therapeutic implications
      • Imatinib: ineffective if mutation is present
      • Dasatinib and Nilotinib: uncertain clinical efficacy
  • Not detected: no detectable KIT (D816V) point mutation

Limitations

  • Mutations other than the D816V mutation are not detected, including other D816 variants
  • Mutations below analytical sensitivity will not be detected

Gastrointestinal Stromal Tumor Mutations and KIT Mutations, Melanoma

Analytical Sensitivity
Variant Class No. Variant Tested Positive Percent Agreement (PPA) PPA, 95% Tolerance at 95% Reliability

SNV

177

100%

98.9-100.0%

MNVs

42

95%

85.6-99.0%

Small insertions and duplicationsa

42

100%

95.6-100.0%

Medium insertions and duplicationsb

10

100%

82.9-100.0%

Large insertionsc

1

100%

22.9%-100.0%

Small deletionsa

80

100%

97.6-100.0%

Medium deletionsb

14

100%

71.2%-99.2%

Large deletionsd

22

64%

42.9%-81.1%

a≤21 bp

b22-60 bp

c≥61 bp and ≤64bp

d≥61 bp and ≤13547bp

bp, base pair; MNVs, multiple nucleotide variants; SNV, single nucleotide variant

Results

  • Detected: KIT mutation detected in exons 9, 11, 13 , 14, 17, 18
  • Detected: PDGFRA mutation detected in exons 12, 14, 18
  • Not detected: no mutations detected in KIT and PDGFRA

Limitations

  • Mutations outside of targeted exons are not detected
  • Test alone cannot be used for diagnosis of malignancy
  • Variants below the limit of detection (LOD) of 5% VAF may not be detected
  • 10 ng input DNA from extracted tissue sample is minimally required, but 50 ng input DNA is recommended for optimal results
  • Large variants (>60bp) may not be detected
  • Not intended to detect minimal residual disease
  • Does not distinguish between somatic and germline variants

References

Additional Resources