Capillary Electrophoresis
Droplet Digital PCR (ddPCR)
- 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
Massively Parallel Sequencing
- Detects activating mutations in KIT and PDGFRA
- Predicts response to targeted therapy
Massively Parallel Sequencing
- Detects activating mutations in KIT and PDGFRA
- Predicts response to targeted therapy
Related Tests
Massively Parallel Sequencing
Use to assess for multiple gene mutations, including substitutions and smaller insertions and deletions, that may have prognostic and/or therapeutic significance
Massively Parallel Sequencing
Use to assess for single gene mutations, including substitutions and smaller insertions and deletions, that may have prognostic and/or therapeutic significance in AML
Fluorescence in situ Hybridization (FISH)
- 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
Fluorescence in situ Hybridization (FISH)
Aids in diagnosis and classification of hematologic neoplasms presenting with prominent eosinophilia
Immunohistochemistry
Initial screening test when GIST or mastocytosis is suspected based on histology and location of tumor
Immunohistochemistry
- 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
Polymerase Chain Reaction/Pyrosequencing
- Detects activating BRAF mutations at codon 600
- Predicts response to targeted therapy in melanomas and colorectal cancers
- Assesses prognosis of certain thyroid cancers
Polymerase Chain Reaction
- 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)
- Adults
- 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
- KIT mutations present in ~85% of cases
- 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
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
-
WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th Ed.
Swerdlow S, Campo E, Harris N, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. International Agency for Research on Cancer; 2008.
22619125
Grossmann AH, Grossmann KF, Wallander ML. Molecular testing in malignant melanoma. Diagn Cytopathol. 2012;40(6):503-510.
NCCN - Acute Myeloid Leukemia
National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Acute myeloid leukemia. Version 3.2020. [Last update: Dec 2019; Accessed: Sep 2020]
NCCN - Cutaneous Melanoma Version 2.2021
National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Cutaneous melanoma. Version 2.2021. [Last update: Feb 2021; Accessed: Apr 2021]
NCCN - gastrointestinal stromal tumors v1.2021
National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Gastrointestinal stromal tumors (GISTs). Version 1.2021. [Last update: Oct 2020; Accessed: April 2021]
NCCN - Systemic Mastocytosis V1 2020
National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Systemic mastocytosis, version 1.2020. [Published: May 2020; Accessed: Jul 2020]
Prognostication in CBF AML