UNIVERSITY OF MALTA
LIFE SCIENCE RESEARCH SEMINARS
Web: http://www.um.edu.mt/events/scisem/
Email: scisem@um.edu.mt
Abstract form
Title: Mutational analysis of c-KIT receptor in gastrointestinal stromal
tumours.
Presenter: Charlene Busuttil
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Abstract
Histopathology Laboratory – Mater Dei Hospital
25456437
charlene.busuttil@gmail.com
24th February, 2014
.
Introduction: The pathogenesis of approximately 80% of gastrointestinal stromal tumours
(GISTs) is associated with activating mutations of the proto-oncogene tyrosine-protein
kinase KIT (c-KIT). Activating mutations are also found in the homologous tyrosine-protein
kinase platelet-derived growth factor receptor α (PDGFRA). Accurate diagnosis of GIST has
become very important since the availability of targeted therapy with tyrosine kinase
inhibitors, such as imatinib. The routine work-up for GIST diagnosis includes
immunohistochemistry for CD117 (c-KIT receptor), as it is estimated that 95% of GIST
cases show positive immunoreactivity. However, it can be observed that the routinely used
immunohistochemical analysis does not provide complete sensitivity for GIST diagnosis, as
there are nearly 5% of GISTs that are negative for c-KIT immunohistochemistry.
Aim: To identify c-KIT mutations present in GIST cases diagnosed locally and to establish
a fast and cost-effective method of testing that includes the combination of 4 exons (c-KIT
exons 9, 11, 13 and 17) to perform a single sequencing reaction.
Methodology: Fifty-two formalin-fixed, paraffin-embedded sections (FFPE) sections from
47 GIST patients diagnosed in the last 14 years were retrieved from the archives of the
Histology Section at the Pathology Department (Mater Dei Hospital). Haematoxylin and
eosin staining and immunhistochemical staining of CD117 were performed on serial
sections to identify tumoural areas. Tumoural tissue was laser microdissected and DNA was
isolated following standard protocols. Polymerase chain reaction (PCR) was used to amplify
exons 9, 11, 13, and 17 of the c-KIT gene followed by sequencing analysis. In addition, high
resolution melting (HRM) analysis was optimized for the detection of the missense mutation
Val560Asp found in exon 11. Ninety-six cord blood samples (assumed to belong to healthy
individuals) were subjected to HRM analysis. Moreover, a novel technique of sequential
amplification of target DNA was optimised by designing primers that enabled fusion of the
amplified fragments (exon 9+11+13+17) ultimately allowing sequencing of the concatamer
in a single run.
Results: Positive CD117 immunostaining was present in 94.2% of the tumours. To date,
mutations where identified in 37 samples (71.1%). All of these mutations were found in cKIT exon 11. The missense mutation Val560Asp was atypically present at a very high
frequency (20/37, 54.1%). HRM analysis results showed that this single nucleotide
substitution is not found in the normal population. Other missense mutations (7/37, 18.9%)
and deletions (10/37, 27%) were also identified. Sequential amplification of target DNA was
successfully performed on 7 samples.
Conclusion: Most mutations were identified in a notable hot spot region in the
juxtamembrane domain of the c-KIT receptor and are known to activate its kinase activity.
Exon 11 mutations are sensitive to imatinib therefore patients harbouring the identified
mutations were eligible for this targeted therapy. Mutational analysis can confirm diagnosis
of GIST especially in CD117-negative suspect cases, can provide prognostic information
and has the ability of predicting therapy outcome.