PDGFRA MUTATION ANALYSIS(AP)
Label Name: PDGFRA
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB9453 
Last Review:  3/17/2017 10:03:49 AM
Specimen Type
  Tissue
Container & Volume
  Age Group   Container   Volume  
  0  - 8 Years CHECK WITH LABORATORY 0.1 ML
  8 Years - 15 Years CHECK WITH LABORATORY 0.1 ML
  15 Years - 18 Years CHECK WITH LABORATORY 0.1 ML
Collection Notes
  All:
  • 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
PDGFRA
Mutation not detected
Methodology
  This assay uses traditional end-point PCR followed by Sanger DNA sequencing to detect mutations within exon 12 and 18 of PDGFRA. 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. Two primer sets are used to amplify exon 12 and exon 18 of the PDGFRA gene. 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 Biosystem). 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 5.2 and SeqScape software v2.6. Sequences are compared to the reference DNA sequence (GenBank Accession: NG_009250.1) for the PDGFRA gene.


   
   
Clinical Significance and Interpretive Data
    Mutations in the PDGFRA gene associated with gastrointestinal stromal tumors (GISTS) result in a signaling pathway constitutively activated producing cell proliferation, survival and tumor formation. GISTS are the most common mesenchymal tumor in the GI tract. It is very common for the diagnosis of GISTs to involve a screen for KIT mutations because they occur most often. However, 7% of GISTs carry mutations in the platelet-derived growth receptor ⍺ (PDGFRA) gene, a mutation that is mutually exclusive to KIT. GIST associated mutations found in PDGFRA are most prevalent in exons 12 and 18. The most common mutations appear in exon 18 and the other prevalent mutation is V561D, encoded in exon 12.The drug Imatinib is a small molecule kinase inhibitor that selectively targets platelet derived growth factor receptor tyrosine kinases. The sensitivity of GISTS to imatinib fluctuates depending on the exonic region that the mutation is located. Patients with exon 18 D842V mutations have been reported to be resistant to imatinib and typically don’t respond well to therapy. This mutation also has shown in my studies to be resistant to other tyrosine kinase inhibitors. The rarer exon 12 V561D mutation is located in the juxtamembrane domain and responds better to nilotinib and imatinib.

REFERENCES:
Heinrich MC, Corless CL, Duensing A, McGreevey L, Chen CJ, Joseph N, Singer S, Griffith DJ, Haley A, Town A, Demetri GD, Fletcher CD, Fletcher JA. PDGFRA activating mutations in gastrointestinal stromal tumors. Science. 2003 Jan 31;299(5607):708-10. Epub 2003 Jan 9. PubMed citation.

Lasota J, Miettinen M (2008). Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal tumours. Histopathology 53(3):245-66.

Corless C, Schroeder A, et al: PDGFRA Mutations in Gastrointestinal Stromal Tumors: Frequency, Spectrum and In Vitro Sensitivity to Imatinib. Journal of Clinical Oncology Vol 23, 23 August 2005.

Chompret, A.; Kannengiesser, C.; Barrois, M.; Terrier, P.; Dahan, P.; Tursz, T.; Lenoir, G.M.; Bressac-De Paillerets, B. PDGFRA germline mutation in a family with multiple cases of gastrointestinal stromal tumor. Gastroenterology 2004, 126, 318–321.

Weisberg E, Wright RD, Jiang J et al. Effects of PKC412, nilotinib, and imatinib against GIST-associated PDGFRA mutants with differential imatinib sensitivity. Gastroenterology 2006; 131; 1734–1742.

   
Indications
    The Platelet-derived growth factor receptor alpha (PDGFRA) gene maps to 4q12, has 23 exons and is adjacent to the KIT locus. The receptor tyrosine kinases are responsible for signal transduction between cells. The PDGFRA protein found to attach to the cell membranes of the protein platelet derived growth factor which activates the PDGFRA protein that activates other proteins in the cell through phosphorylation. This process leads to the activation of a series of proteins in multiple signaling pathways which control cellular processes such as cell growth, division, and survival.Ή Lack of control in these pathways can lead to uncontrolled cell growth and tumor development. The most common tumors in the mesenchymal tissue of the gastrointestinal tract are gastrointestinal stromal tumors or GISTs. It is very common for the diagnosis of GISTs to involve a screen for KIT mutations because they occur most often. However, 7% of GISTs carry mutations in the platelet-derived growth receptor ⍺ (PDGFRA) gene, a mutation that is mutually exclusive to KIT. GIST associated mutations found in PDGFRA are most prevalent in exons 12 and 18. The most common mutation in exon 18 and the other prevalent mutation is V561D, encoded in exon 12. Due to KIT and PDGFRA mutations being exclusive and the clinical implications of PDGFRA on imatinib sensitivity, PDGFRA mutation analysis is frequently being ordered as a reflex test when the GIST patient tested negative for the KIT mutation.
   
Limitations
    • The sensitivity of DNA sequencing is high for the detection of nucleotide base changes and small deletions and insertions in the regions analyzed.
• Only exons 12 & 18 are examined in the PDGFRA gene.
• Changes in other regions of the gene are not examined, and mutations that fall in these regions will not be detected.
• Mutations in genes other than PDGFRA will not be identified.
• Large deletions, duplications, multiple exon insertions, sequence alterations adversely affected primer binding, and complete deletion of one allele may not be identified using these methods.
• This assay may not detect an acquired mutation which is present below the 15% tumor content detection limit (i.e., mutant cell population containing at least 15% of total cells from the genomic DNA is extracted for testing).
• Clinicians should correlate results with clinical findings

   
Test Synonyms
  Synonym(s): KIT, GIST
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

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