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Annals of Oncology Advance Access originally published online on January 10, 2008
Annals of Oncology 2008 19(4):706-710; doi:10.1093/annonc/mdm503
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© The Author 2008. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

gastrointestinal tumors

KIT and PDGFR{alpha} mutations in 104 patients with gastrointestinal stromal tumors (GISTs): a population-based study

C. Braconi1,7,*, R. Bracci1,3, I. Bearzi2, F. Bianchi1, A. Costagliola2, R. Catalani1, A. Mandolesi2, R. Ranaldi2, E. Galizia1, S. Cascinu3, G. Rossi4, L. Giustini5, L. Latini6, N. Valeri8,9 and R. Cellerino1

1 Centro Regionale di Genetica Oncologica, Oncologia Medica, Ancona
2 Anatomia ed Istologia Patologica, Università Politecnica delle Marche, Ancona
3 Clinica di Oncologia Medica, Ospedali Riuniti, Ancona
4 Oncologia Medica, Ospedale Murri, Jesi
5 Oncologia Medica, Ospedale di Fermo, Fermo
6 Oncologia Medica, Ospedale di Macerata, Macerata, Italy
7 Department of Internal Medicine, Ohio State University, Columbus, OH, USA
8 Scuola di Specializzazione in Oncologia, Università Politecnica delle Marche, Ancona, Italy
9 Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, OH, USA

* Correspondence to: Dr C. Braconi, Centro Regionale di Genetica Oncologica, Oncologia Medica, Università Politecnica delle Marche, via Tronto 1, 60126 Ancona, Italy. Tel: +39-71-2205161; Fax: +39-71-2206191; E-mail: chiarabraconi{at}tiscali.it


   Abstract
Top
 Abstract
introduction
 materials and methods
 results
 conclusions
 Acknowledgements
 References
 
Background: The prognostic significance of KIT or platelet-derived growth factor receptor {alpha} (PDGFR{alpha}) mutations in gastrointestinal stromal tumors (GISTs) is still controversial.

Patients and methods: In all, 104 patients were diagnosed with GISTs by KIT immunoreactivity; tumor DNA was sequenced for the presence of mutations in KIT exons 9, 11, 13 and 17 and in PDGFR{alpha} exons 12 and 18. Disease-free survival (DFS) was analyzed in 85 radically resected patients.

Results: KIT mutations occurred in exon 11 (69), in exon 9 (11) and in exon 17 (1). PDGFR{alpha} mutations were detected in exon 18 (10) and in exon 12 (3). Ten GISTs were wild type. Exon 11 mutations were as follows: deletions in 42 cases and point mutations in 20 cases and insertions and duplications, respectively, in 2 and 5 cases. A better trend in DFS was evident for duplicated and point-mutated exon 11 KIT GISTs. There was a significant association between PDGFR{alpha} mutations, gastric location and lower mitotic index. Moreover, PDGFR{alpha}-mutated GISTs seemed to have a better outcome.

Conclusions: Point mutations and duplications in KIT exon 11 are associated with a better clinical trend in DFS. PDGFR{alpha}-mutated GISTs are preferentially localized in the stomach and seem to have a favorable clinical behavior.

Key words: GISTs, KIT, mutations, PDGFR{alpha}, prognosis


   introduction
Top
 Abstract
 introduction
materials and methods
 results
 conclusions
 Acknowledgements
 References
 
Gastrointestinal stromal tumors (GISTs) are rare neoplasms, whose incidence is ~0.68–1.5 cases/100000/year [1]. Ninety percent of GISTs are positive for KIT and 10% are positive for platelet-derived growth factor receptor {alpha} (PDGFR{alpha}). KIT and PDGFR{alpha} are two transmembrane receptors, whose intracellular region is subdivided into a juxtamembrane domain (JMD) and two tyrosin kinase domains [2]. Mutations in these genes, resulting in constitutively phosphorylated and activated receptors, are responsible for 90% of GISTs. Most of the mutations (65%) occur in KIT exon 11, encoding for the JMD. Only 10% of GISTs harbor mutations in KIT exon 9, encoding for the extracellular region. Mutations in exons 13 and 17 of KIT, which encode, respectively, for tyrosin kinase domains 1 and 2, are sporadic. PDGFR{alpha} is involved in 7%–10% of GISTs and mutations occur preferentially in exon 18 (kinase domain 2), rarely in exon 12 (JMD) and sporadically in exon 14 (kinase domain 1).

The prognostic role of different types of mutations has been studied. Exon 9 KIT mutations seem to define a particular subgroup of GISTs with unfavorable outcome [3]. No definitive prognostic value for exon 11 KIT mutations was found [4]. Singer et al. indicated a better trend in survival for patients with exon 11 point-mutated GISTs, while Heinrich et al. [5] and Kim et al. [6] found no difference between exon 11 deletions and point mutations. The aim of this study was to evaluate the prognostic impact of KIT and PDGFR{alpha} mutations in a sample of GISTs observed in the clinical practice of our region.


   materials and methods
Top
 Abstract
 introduction
 materials and methods
results
 conclusions
 Acknowledgements
 References
 
histopathology and immunohistochemical review
Tumor specimens included in this study were retrieved among patients who received diagnosis of primary GISTs at Pathology Department of Ancona from November 1987 to June 2006. An immunohistochemical reaction for KIT (anti-CD117) was carried out in every case with a polyclonal rabbit antibody (DAKO Corp., Carpinteria, CA). GISTs were assigned a class of risk of malignant behavior (high, intermediate, low and very low) according to the indications of literature [7]. In our analysis, we grouped together patients with GISTs belonging to very low and low-risk classes.

molecular analysis
A molecular analysis was carried out for all cases. DNA was extracted from formalin fixed and paraffin-embedded tumor tissue samples. Polymerase chain reaction (PCR) was carried out in order to amplify KIT exons (9, 11, 13 and 17) and PDGFR{alpha} exons (12 and 18). All PCR conditions and primer sequences are available to the corresponding author. We analyzed PCR products by direct sequencing with an ABI 310 Genetic Analyzer (Applied Biosystem, Foster City, CA) according to the manufacturer's recommendations.

Exons were evaluated in the following sequence: KIT exon 11, exon 9, PDGFR{alpha} exon 18, exon 12, KIT exon 13 and exon 17. Each sample was studied for the following exon if no mutations were previously found. All the mutations found were confirmed by a new PCR and a new sequencing analysis. Our results were compared with normal sequence available on Gene ID 3815 (KIT) and Gene ID 5156 (PDGFR{alpha}).

clinical follow-up and statistical analysis
Disease-free survival (DFS) was used as an end point of the study. Disease recurrence was defined as the presence of peritoneal or liver disease. The following clinicopathological variables were studied: age, sex, tumor size, location, mitotic index, class of risk, morphological subtype and type of mutations. The association of these variables with the presence and the type of mutation was tested using {chi}2 test. The log-rank test was used to assess the relation of individual clinicopathological and molecular variables to DFS. P value <0.05 was considered to be statistically significant. No patients received adjuvant or neo-adjuvant therapy.


   results
Top
 Abstract
 introduction
 materials and methods
 results
conclusions
 Acknowledgements
 References
 
patient demographics and tumor characteristics
We analyzed 104 patients with GISTs. Fifty-eight patients were men and 46 were women. Median age at diagnosis was 63 years (range 30–91) (Table 1).


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  		Table 1. Characteristics of tumors

 
molecular analysis
Exon 11 KIT mutations (69) were as follows: deletions in 42 cases (61%), single point mutations in 20 cases (29%) and insertions and duplications in the reminder 10% (Table 1). Most of the exon 11 deletions occurred in codons 557–561, even though codons 555–556 and 562–572 were also frequently involved. Point mutations occurred preferentially in codon 559. The five exon 11 duplications were localized in 3'-terminal codons (571–588).

Among the five exon 11 duplicated GISTs, four were gastric (two epithelioid and two spindle) and one (mixed) was intestinal. All these GISTs had a low mitotic count. All these old female patients are still alive free of recurrence, except one who died for other reasons free of disease.

Among the 11 patients with exon 9 KIT-mutated GISTs, 9 had a tumor of the small bowel and 2 of the retroperitoneum. Seven tumors were high, three intermediate and one low risk. A significant association was found between exon 9 KIT-mutated GIST and intestinal site (P = 0.0003). All exon 9 mutations were duplication of six nucleotides (codons 502–503).

All 13 PDGFR{alpha}-mutated GISTs developed in the stomach. The three exon 12 PDGFR{alpha} tumors were spindle cell and two of them were very low-risk tumors. Among 10 exon 18-mutated GISTs, 3 were low risk, 3 intermediate and 4 high-risk tumors; none of them was spindle cell, while most of them (60%) were epithelioid. All exon 18 PDGFR{alpha} mutations occurred in codon 842. PDGFR{alpha}-mutated GIST correlated with low mitotic index (<5/50HPF) (P = 0.005), epithelioid histology (P = 0.05) and gastric location (P = 0.0003).

Among wild-type GISTs (10), there were four (40%) high risk, three (30%) intermediate and three (30%) low-risk tumors; the most frequent sites were stomach (seven) and small bowel (three).

clinical data
Clinical information was detected with the collaboration of different Medical Oncology Units of Marche Region, Italy. At a median follow-up of 3.7 years, 27 patients (26%) died because of GIST and 14 patients (13%) died for other reasons free of disease. Sixty-three patients (61%) are still alive.

Nineteen patients (18%) were diagnosed with metastatic disease (Table 1). In this subgroup of patients, median overall survival was 2.1 years. It was not correlated with the sites of metastatic disease.

Eighty-five patients underwent radical surgery for localized GIST (Table 1). Twenty-three (27%) patients relapsed; 22 of them had high-risk GISTs, while 1 patient had intermediate-risk GIST. Among high-risk relapsed GISTs (22), 77% (17) had exon 11 KIT deletions, 13% (3) had exon 9 KIT mutations, 5% (1) was wild type and 1 (5%) was exon 18 PDGFR{alpha} mutated.

We evaluated DFS in the 85 patients with localized disease at diagnosis, who were radically resected. In this subgroup of patients, median DFS was 13.6 years. At univariate analysis, size of tumor (≥10 versus <10 cm; P = 0.0004), mitotic count (≥10 versus <10 mitosis/50HPF; P < 0.0001) and class of risk (P < 0.0001) were prognostic factors. DFS was not influenced by the histotype. Mutational assessment did not correlate with DFS (Figure 1). There were no significant differences comparing subgroups of patients having GISTs with different mutations (Figure 2). Among patients with exon 11 KIT-mutated GISTs, the different types of mutation did not correlate with risk of relapse (Figure 3A). A favorable trend in DFS, however, was observed in patients with exon 11 point mutations (P = 0.1), even when compared with all other mutations (P = 0.3) (Figure 3B). The numeric imbalance between the two arms might be responsible for the missed achievement of a statistical significance.


Figure 1
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  		Figure 1. Correlation between disease-free survival (DFS) and different types of mutations. KIT exon 11 mutations: 54 patients (median DFS 10.6 years). KIT exon 9 mutations: 8 patients (median DFS 10.2 years). Platelet-derived growth factor receptor {alpha} (PDGFR{alpha}) mutations: 13 patients (median DFS not reached). Wild type: 9 patients (median DFS not reached). The only one case with KIT exon 17 mutation was not included in the analysis. (P = 0.2).

 

Figure 2
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  		Figure 2. (A) Comparison among KIT exon 11 mutations, wild type and all other mutations. KIT exon 11 mutations: 54 patients [median disease-free survival (DFS) 10.6 years]. All other mutations: 22 patients (median DFS 10.2 years). Wild type: 9 patients (median DFS not reached) (P = 0.3). (B) Comparison among KIT exon 9 mutations, wild type and all other mutations. KIT exon 9 mutations: 8 patients (median DFS 10.2 years). All other mutations: 68 patients (median DFS 13.6 years). Wild type: 9 patients (median DFS not reached) (P = 0.3). (C) Comparison among platelet-derived growth factor receptor {alpha} (PDGFR{alpha}) mutations, wild type and all other mutations. PDGFR{alpha} mutations: 13 patients (median DFS not reached). All other mutations: 63 patients (median DFS 10.2 years). Wild type: 9 patients (median DFS not reached) (P = 0.2).

 

Figure 3
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  		Figure 3. (A) Comparison among different types of mutations within KIT exon 11. Duplications: 5 patients [median disease-free survival (DFS) not reached]. Insertions: 2 patients [median disease-free survival (DFS) not reached]. Point mutations: 16 patients (median DFS not reached). Deletions: 32 patients (median DFS 10.6 years). Insertions and duplications curves overlap (P = 0.1). (B) Comparison between KIT exon 11 point mutations and all other types of mutations. KIT exon 11 point mutations: 16 patients (median DFS not reached). All other mutations: 60 (median DFS 10.6 years). Wild type tumors have not been included in the analysis (P = 0.3).

 

   conclusions
Top
 Abstract
 introduction
 materials and methods
 results
 conclusions
Acknowledgements
 References
 
In GISTs, the ligand-independent stimulation of receptors results in cell proliferation and survival. This activation is due to mutations in genes encoding mainly for KIT and more rarely for PDGFR{alpha}. We found mutation of KIT in 79% of the entire sample. Sixty-seven percent of GISTs harbored mutations in exon 11 of KIT. Some authors indicate that GISTs with exon 11 KIT deletions have a more aggressive behavior than GISTs with exon 11 KIT insertions or point mutations [8–11]. We did not find any significant correlation among the subtypes of exon 11 KIT mutations and DFS, even though there is a positive trend (P = 0.1) for those patients with GISTs harboring exon 11 KIT point mutations compared with all other mutations.

Exon 11 insertions and duplications are very rare. According to Antonescu et al. [3], duplication identifies a peculiar subgroup of old female patients with gastric indolent GISTs. We also observed an association among these GISTs and gastric location, older age, female sex and favorable clinical behavior.

Less frequently KIT is mutated in the extracellular region coded by exon 9. We found this mutation in 11% of GISTs. Both Lasota et al. [12] and Antonescu et al. [3] identified a prognostic value for exon 9 KIT mutations. We confirmed the association of this mutation with preferential intestinal location (P = 0.0003), but we did not demonstrate any significant correlation with DFS, likely due to the imbalance between the two arms of the analysis (Figure 2B).

Debiec-Rychter et al. [13] and Antonescu et al. [3] noticed an association between gastric epithelioid GISTs and the absence of KIT mutations. In our series, gastric epithelioid GISTs were wild type in four cases and exon 11 KIT mutated in six cases. Besides, we observed a high number of gastric epithelioid GISTs with PDGFR{alpha} mutations (6 of 10), which might have been classified as wild-type GISTs in the previous studies. A significant association was found between PDGFR{alpha}-mutated GISTs and gastric location (p.0003) and epithelioid histology (p.005), as recently found by Tornillo et al. [10]. Moreover, tumors with exon 18 PDGFR{alpha} mutations seemed to have a favorable clinical behavior. Also the other three tumors harboring exon 18 PDGFR{alpha} mutations, but mixed rather than epithelioid, were gastric GISTs with a low tendency to relapse. In a univariate analysis, the mutation in exon 18 PDGFR{alpha}, however, did not correlate with DFS, but this, once again, could be ascribed to the small size of this group.

The prognostic role of wild-type GISTs is still a debate. Kim et al. [6] observed a better 5 years DFS for patients with wild-type tumors rather than patients with mutated GISTs.

In the present study, a statistical significant difference in DFS was not detected between these two classes of GISTs, although we could recognize a better clinical trend in DFS for patients affected by GISTs without mutations (Figure 2). Only when compared with PDGFR{alpha}-mutated GISTs, wild-type tumors did not seem to show a better behavior.

In conclusion, our study of 104 patients with GISTs confirms the prognostic value of factors such as size, mitotic count and class of risk. The majority of GISTs showed KIT mutations (79%). Patients with exon 11-mutated GISTs were not characterized by a different clinical outcome, even though point mutations and duplications seem to define subgroups of patients with better prognosis. Exon 9 KIT-mutated GISTs occurred preferentially in small bowel. PDGFR{alpha}-mutated GISTs were all gastric tumors with a non-aggressive behavior. Wild-type GISTs had no significant different outcome from others, although a favorable trend could be indicated.


   Acknowledgements
Top
 Abstract
 introduction
 materials and methods
 results
 conclusions
 Acknowledgements
References
 
We would like to thank E. T. Menichetti, R. Mattioli, R. R. Silva and G. Catalano for their contribution to the collection of clinical data.

Received for publication August 29, 2007. Revision received September 25, 2007. Accepted for publication September 28, 2007.


   References
Top
 Abstract
 introduction
 materials and methods
 results
 conclusions
 Acknowledgements
 References
 
1. Perez EA, Livingstone AS, Franceschi D, et al. Current incidence and outcomes of gastrointestinal mesenchymal tumors including gastrointestinal stromal tumors. J Am Coll Surg (2006) 202:623–629.[CrossRef][Web of Science][Medline]

2. Lev S, Blechman J, Nishikawa S, et al. Interspecies molecular chimeras of kit help define the binding site of the stem cell factor. Mol Cell Biol (1993) 13:2224–2234.[Abstract/Free Full Text]

3. Antonescu CR, Sommer G, Sarran L, et al. Association of KIT exon 9 mutations with nongastric primary site and aggressive behavior: KIT mutation analysis and clinical correlates of 120 gastrointestinal stromal tumors. Clin Cancer Res (2003) 9:3329–3337.[Abstract/Free Full Text]

4. Singer S, Rubin BP, Lux ML, et al. Prognostic value of KIT mutation type, mitotic activity, and histologic subtype in gastrointestinal stromal tumors. J Clin Oncol (2002) 20:3898–3905.[Abstract/Free Full Text]

5. Heinrich MC, Corless CL, Blanke CD, et al. Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J Clin Oncol (2006) 24:4764–4774.[Abstract/Free Full Text]

6. Kim TW, Lee H, Kang YK, et al. Prognostic significance of c-kit mutation in localized gastrointestinal stromal tumors. Clin Cancer Res (2004) 10:3076–3081.[Abstract/Free Full Text]

7. Fletcher CD, Berman JJ, Corless C, et al. Diagnosis of gastrointestinal stromal tumors: a consensus approach. Hum Pathol (2002) 33:459–465.[CrossRef][Web of Science][Medline]

8. De Matteo RP, Lewis JJ, Leung D, et al. Two hundred gastrointestinal stromal tumors: recurrence patterns and prognostic factors for survival. Ann Surg (2000) 231:51–58.[CrossRef][Web of Science][Medline]

9. Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J Clin Oncol (2004) 22:3813–3825.[Abstract/Free Full Text]

10. Tornillo L, Terracciano LM. An update on molecular genetics of gastrointestinal stromal tumours. J Clin Pathol (2006) 59:557–563.[Abstract/Free Full Text]

11. Martin J, Poveda A, Llombart-Bosch A, et al. Deletions affecting codons 557-558 of the c-KIT gene indicate a poor prognosis in patients with completely resected gastrointestinal stromal tumors: a study by the Spanish Group for Sarcoma Research (GEIS). J Clin Oncol (2005) 23:6190–6198.[Abstract/Free Full Text]

12. Lasota J, Wozniak A, Sarlomo-Rikala M, et al. Mutations in exons 9 and 13 of KIT gene are rare events in gastrointestinal stromal tumors. A study of 200 cases. Am J Pathol (2000) 157:1091–1095.[Abstract/Free Full Text]

13. Debiec-Rychter M, Lasota J, Sarlomo-Rikama M, et al. Chromosomal aberrations in malignant gastro-intestinal stromal tumors: correlation with c-kit gene mutation. Cancer Genet Cytogenet (2001) 128:24–30.[CrossRef][Web of Science][Medline]


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