PhD of Costagliola Antonello
PATHOGENESIS AND MOLECULAR ANALYSIS
OF C-KIT AND PDGFR GENES
IN GASTROINTESTINAL STROMAL TUMORS (GISTs)
ABSTRACT
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the
gastrointestinal tract and rising from interstitial cells of Cajal (ICC) or from stem cells with the
capacity to differentiate towards interstitial cells of Cajal. Most GISTs have activating mutation in
the c-kit receptor tyrosine kinase (69,5%) and the 50% of Gastrointestinal stromal tumors that
lacking kit mutation, have intragenic activation in the related receptor tyrosine kinase, Plateledderived growth factor receptor A (PDGFR). Kit and PDGFRA have approxymately 35%
homology, and belong to the PDGFR subfamily of receptor tyrosine kinases, which are involved
in the regulation of cell growth, proliferation, adhesion, migration, differentiation and apoptosis.
The downstream signalling consequences of various kit and PDGFR mutations in GISTs are
similar, as are the additional cytogenetic changes associated with tumor progression. Kit and
PDGFR mutations appear to be alternative and mutually exclusive oncogenic mechanisms in
GISTs and these mutations have similar biological consequences.
Methods: 131 tumor samples obtained betwen 1984-2006 have retrieved from the archives of
the Anatomy Pathology Institute in Ancona (Italy). Cases are classificated based on ‘Consensus
Conference of Bethesda’ (2001) for aggressive behavior in four risk’s grade: high, intermediate,
low and very low risk. Tumor tissue are microdissected from 5-10 micron serial section. Extraction
of genomic DNA are performed by proteinase k digestion, followed by ethanol precipitation using
standard procedures. The samples are evalueted for mutations by PCR amplification and direct
sequencing. Cases negative for kit exon 9 and 11 mutations are evaluated for PDGFRA exon 12 and
18 mutations.
Result and Discussion: Kit or PDGFRA mutations are detected in 91 (69.5%) and 14 of 28
(50%) of GISTs respectively. Most kit mutations involved exon 11 (77 cases, 84.6%), followed by
exon 9 (14 cases, 15.4%). The PDGFRA mutations mostly affected exon 18 (11 cases of 28,
39,3%), followed by exon 12 (3 cases of 28, 10,7%). The detected mutations within kit exon 11 are
heterogeneous and consist of 45 simple deletions, 23 point mutations, 8 internal tandem
duplications and one insertion. Most kit exon 11 mutations are located at the 5’ end of exon, while
all internal tandem duplications (5-45 amino acids) are clustered inthe 3’ part. The most common
genetic alteration in in kit exon 11 are the 13 amino acid substitutions, followed by 9 deletions of
codons 557-558. The 10 cases with kit exon 9 mutations showed an identical 6 bp insertion,
resulting in a tandem duplication of the amino acids Ala502 and Tyr503. PDGFR mutations
affecting exon 18 consisted of a D842V substitution (10 cases) and 1 deletion (codon 843).
Furthermore, three tumors harboured a V561D substitution in PDGFR exon 12 gene.
With respect to the primary site, kit exon 9 mutations are associated with GISTs of intestinal origin,
whereas PDGFR mutations are largely confined to tumors of gastric origin (100% of cases). With
regard to the histological phenotype, kit mutations are seen especially in spindle (79,7%) and mixed
(70.3%) cells GISTs. Applyng the risk assesment of primary tumors according to Fletcher et al, the
75% of ‘high risk’ tumors are kit mutated, and the 50% have metastasys. Either GISTs with
‘intermediate’ (64%) and ‘low’ (60%) risk have kit mutation. Furthermore, the presence of any kit
mutations alone are significantly associated with high risk/malignant GISTs. For these motive,
mutations of kit or PDGFR gene don’t have prognostic value, but molecular study is important for
differentiate the type and the exon mutation. In fact the exon 9 mutation is often associated to high
risk. In addition GISTs with kit mutations have metastasied and/or relapsed most frequently than
wilde-type tumors.
Finally, Imatinib Mesylate (Gleevec) – a selective inhibitor of kit and PDGFR tyrosine kinases –
has a high response rate in patiens with advanced GISTs, witch are largely radiotherapy and
chemotherapy resistant. Several studies have evidenced that the type and location of kit or PDGFR
mutations in GISTs predicts the response to imatinib treatment: tumors harbouring a kit exon 11
mutation have a significantly higher response rate and a longer event free and overall survival
compared with those containing a kit exon 9 or no mutation. Tumors with the PDGFR exon 18
mutation D842V showed no response to imatinib treatment, whereas patients with GISTs
harbouring other PDGFR mutations archived partial remission.
Our study suggests that the type and location of receptor tyrosine kinase mutations in GISTs may be
a helpful additional parameter in determining the biological profile of these tumors, for a better
treatment.