Skip Navigation


Human Molecular Genetics Advance Access originally published online on February 6, 2006
Human Molecular Genetics 2006 15(6):1015-1023; doi:10.1093/hmg/ddl016
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
15/6/1015    most recent
ddl016v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (16)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Maertens, O.
Right arrow Articles by Legius, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maertens, O.
Right arrow Articles by Legius, E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Molecular pathogenesis of multiple gastrointestinal stromal tumors in NF1 patients

Ophélia Maertens1,{dagger}, Hans Prenen2,{dagger}, Maria Debiec-Rychter3, Agnieszka Wozniak3,6, Raf Sciot4, Patrick Pauwels7, Ivo De Wever5, Joris R. Vermeesch3, Thomas de Raedt3, Anne De Paepe1, Frank Speleman1, Allan van Oosterom2, Ludwine Messiaen1,8 and Eric Legius3,*

1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 2Department of Clinical Oncology, 3Department of Human Genetics, 4Department of Pathology and 5Oncological Surgery, Catholic University Leuven, Leuven, Belgium, 6Department of Biology and Genetics, Medical University of Gdansk, Gdansk, Poland, 7Department of Pathology, University Hospital Maastricht, Maastricht, The Netherlands and 8Department of Genetics, University of Alabama, Birmingham, AL, USA

* To whom correspondence should be addressed at: Department of Human Genetics, Catholic University Leuven, Herestraat 49, B-3000 Leuven, Belgium. Tel: +32 16345903; Fax: +32 16346051; Email: eric.legius{at}uz.kuleuven.ac.be

Received October 24, 2005; Revised January 18, 2006; Accepted February 1, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. KIT and PDGFRA activating mutations are the oncogenic mechanisms in most sporadic GISTs. In addition to sporadic occurrences, GISTs are increasingly being recognized in association with neurofibromatosis type 1 (NF1), yet the underlying pathogenic mechanism remains elusive. To gain an insight into the mechanisms underlying GIST formation in NF1 patients, we studied seven GISTs from three NF1 patients with a combination of different techniques: mutation analysis (KIT, PDGFRA and NF1), western blotting, array CGH and ex vivo imatinib response experiments. We demonstrate that (i) the NF1-related GISTs do not have KIT or PDGFRA mutations, (ii) the molecular event underlying GIST development in this patient group is a somatic inactivation of the wild-type NF1 allele in the tumor and (iii) inactivation of neurofibromin is an alternate mechanism to (hyper) activate the MAP-kinase pathway, while the JAK-STAT3 and PI3K-AKT pathways are less activated in NF1-related GIST compared with sporadic GISTs. In conclusion, we report for the first time the molecular pathogenesis of GISTs in NF1 individuals and demonstrate that this type of tumor clearly belongs to the spectrum of clinical symptoms in NF1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract (1Go). They occur predominantly in the stomach (70%) and small intestine (10–20%) and originate from primitive cells with characteristics of the interstitial cells of Cajal (ICCs), the autonomic pacemaker cells which regulate peristalsis in the digestive tract (1Go–3Go). Most sporadic GISTs contain gain-of-function mutations in KIT (80–85%) (4Go,5Go) and strongly express the constitutively activated protein (6Go). Some GISTs contain gain-of-function PDGFRA mutations as an alternate oncogenic mechanism (7Go,8Go). Both receptor tyrosine kinases transduce their signals through the downstream PI3K-AKT (9Go), MAP-kinase (10Go,11Go) and JAK-STAT3 signaling cascades (12Go–16Go). Therapeutic targeting with the tyrosine kinase inhibitor imatinib (Gleevec, Novartis, Basel, Switzerland) shows a clinical benefit in up to 80% of patients with sporadic GIST (17Go,18Go).

GISTs are increasingly recognized in adults with neurofibromatosis type 1 (NF1), an autosomal dominant genetic disorder caused by alterations of the NF1 gene and affecting 1/3500 individuals worldwide. Neurofibromin, the protein encoded by NF1, functions as a GTPase-activating protein for Ras by catalyzing the hydrolysis of active Ras-GTP to an inactive Ras-GDP (19Go–23Go). The primary clinical features of the disease are café-au-lait spots, freckling and benign peripheral nerve sheath tumors or neurofibromas (24Go). Neurofibromin deficiency in neurofibromas results in hyperactivation of the MAP-kinase pathway. On the basis of a single Swedish study of 70 NF1 patients, it is estimated that adults with NF1 might have a risk for GISTs as high as 7% (25Go). GISTs in NF1 patients tend to be multiple and are located predominantly within the small intestine (26Go–28Go). Morphologically and immunohistochemically, GISTs occurring in the NF1 patients are similar to sporadic GISTs (26Go–29Go). Little is known about the molecular basis underlying GIST formation in NF1, and whether there is a difference in the molecular pathogenesis of sporadic and NF1-related GISTs. It remains an intriguing question why NF1 patients are at an increased risk for this type of tumor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Histopathological analysis
The NF1-related intestinal tumors corresponded to bona fide spindle cell type GISTs (Fig. 1A). The tumor cells were strongly positive for CD117 (Fig. 1B and 1C). The mast cells served as positive control for CD117, whereas the other tissue elements of the mucosa and submucosa were negative (Fig. 1B and 1C). There was a variable positivity for alpha smooth muscle actin, CD34 and S-100 protein, whereas desmin was negative.


Figure 0161
View larger version (45K):
[in this window]
[in a new window]
  		Figure 1. Histopathological analysis of NF1-related GISTs. (A) Low power view, showing the spindle cell character of the NF1-related GIST (H&E stain, x200). (B) KIT staining shows strong expression in the tumor (immunoperoxidase stain for CD117, x50). (C) Detail of the mucosa. Only the mast cells are KIT positive (immunoperoxidase stain for CD117, x350).

 
Mutation analysis
KIT/PDGFRA
Analysis of the entire KIT and PDGFRA coding region did not reveal any activating mutation in the NF1-related GIST specimens (Table 1). One single nucleotide polymorphism (SNP) in the 3'-UTR region of KIT (rs2213181, http://www.ncbi.nlm.nih.gov/SNP) was revealed in all GISTs derived from patient NF1-1. In the GIST of patient NF1-3, one silent polymorphism was found in KIT (p.K546K) and five alterations were found in PDGFRA: three silent mutations (p.N204N, p.G313G and p.A603A), one 3'-UTR SNP (rs7680422, http://www.ncbi.nlm.nih.gov/SNP) and one missense mutation [c.1825T>C (p.S478P)]. The missense mutation was not predicted to have an impact on protein activity (Polyphen algorithm, http://tux.embl-heidelberg.de/ramensky) and was found in 19/98 (19%) control individuals. All detected variants were also present in DNA extracted from peripheral white blood cells of the respective patients. The detection of heterozygous polymorphisms indicates the presence of both KIT alleles in one out of seven tumors and both PDGFRA alleles in four out of seven tumors.


View this table:
[in this window]
[in a new window]
  		Table 1. Molecular analysis of seven GISTs derived from three NF1 patients
 
NF1
Mutation screening of peripheral blood revealed NF1 germline mutations in all three patients (Table 1). The somatic alterations in the NF1 gene region detected in six of the seven GISTs (Table 1) were three nonsense mutations, one donor splice site mutation, a multi-exon deletion encompassing NF1 exons 10c–21 and one loss of heterozygosity (LOH) event including the NF1 gene. All NF1 mutations are assumed to have an inactivating effect (Table 1). Analysis of the nonsense mutation p.R2616X in different cell types derived from tumor II of patient NF1-2 demonstrated that the somatic NF1 mutation was exclusively present in the cells cultured with the medium selective for ICC culture and not in fibroblasts grown from the same tumor.

Western blotting
All seven NF1-related tumors showed only rudimentary neurofibromin expression in comparison with the control GIST882 cell line harboring the KIT p.K642E mutation and the sporadic GIST with the KIT p.557–558delWK mutation (Fig. 2). NF1-related GISTs demonstrated normal total KIT expression but low levels of constitutive KIT autophosphorylation in comparison to the control GIST882 and sporadic GIST cells (Table 2). Total MAPK and MAPK phosphorylation was ubiquitously more pronounced in NF1-related GIST tissue. On the basis of the densitometric quantification of autoradiographic signals, the relative p-MAPK expression in NF1-related GIST was 3–18-fold higher than in sporadic GIST (Table 2). Expression of total AKT was variable in NF1-related GISTs and not associated with protein phosphorylation. Moreover, NF1 tumors did not express STAT3 protein.


Figure 0162
View larger version (48K):
[in this window]
[in a new window]
  		Figure 2. Analysis of KIT/PDGFRA downstream signaling pathways in two sporadic [GIST882 cell line (KIT p.K642E) and sporadic GIST (KIT p.557–558delWK)] and seven NF1-related GISTs by western blotting. Equal protein loading was demonstrated with anti-actin staining. On the basis of the densitometric quantification of autoradiographic signals, the relative p-MAPK expression in the NF1-related GISTs was 3–18-fold higher than in the sporadic GIST. Because of short exposure time, phosphorylation of MAPK of GIST882 is less visible.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Relative p-KIT and p-MAPK expression based on densitometric quantification of autoradiographic signals obtained by western blotting (data corresponding to Fig. 2)
 
Array CGH experiments
Array CGH experiments on NF1-related GISTs showed similar losses of chromosome regions as seen in sporadic GISTs (i.e. losses of chromosomes 11, 14, 22 and 1p) in addition to several extra alterations (Table 3). These additional chromosomal abnormalities seem not to be recurrent in the different tested NF1-related GISTs. Array CGH confirmed LOH of the NF1 gene in the large tumor of patient NF1-3 [loss of the clones containing the NF1 gene (RPCI5-926B9 and RP11-229K15)] while no other aberrations were observed (Table 3 and Fig. 3).


View this table:
[in this window]
[in a new window]
  		Table 3. Summary of array CGH experiments on seven GISTs derived from three NF1 patients
 

Figure 0163
View larger version (32K):
[in this window]
[in a new window]
  		Figure 3. Array CGH profiles of seven NF1-related GISTs. Profiles are composed of numerically ordered autosomes (1Go–22Go) followed by X and Y. Arrows below and above profiles mark losses and gains, respectively.

 
Phenotype of cultured cells
After 3 days in culture, the primary cells of tumor III from patient NF1-2 exhibited spindle shape type of growth and 95% were KIT immunopositive. The Ki-67 labeling index was 0.5 and 8.7% in ligand unstimulated and ligand stimulated cultures, respectively (Fig. 4).


Figure 0164
View larger version (61K):
[in this window]
[in a new window]
  		Figure 4. Phenotype of cultured cells. (A) After 3 days in culture, the primary cells of tumor III from patient NF1-2 exhibited spindle shape type of growth and 95% were KIT immunopositive. (B) The Ki-67 labeling index was 0.5 and 8.7% in ligand unstimulated and ligand stimulated cultures, respectively.

 
Imatinib assay
As shown in Figure 5, KIT phosphorylation of primary NF1-related GIST cells seeded in culture is ligand-dependent as shown by very limited KIT phosphorylation in the absence of stem cell factor (SCF). SCF-dependent KIT phosphorylation of NF1-related GIST cells is totally inhibited by exposure to imatinib at concentration of 0.5 µM. The same inhibition is observed in the control autophosphorylated GIST882 cell line harboring the KIT p.K642E mutation. The level of MAPK phosphorylation of primary NF1-related GIST cells was moderately decreased after exposure to imatinib but the inhibition was neither complete nor dose-dependent. Imatinib decreased in a dose-dependent way the level of MAPK autophosphorylation at concentrations lower than 1.0 µM in the GIST882 cell line but not at any tested doses in control fibroblasts. The relative p-KIT and p-MAPK expressions, based on the densitometric quantification of autoradiographic signals, are shown in Table 4.


Figure 0165
View larger version (33K):
[in this window]
[in a new window]
  		Figure 5. Ex vivo effect of imatinib on the phosphorylation of KIT Y703 and MAPK T202/Y204 residues in control GIST882 cell line (KIT p.K642E), primary NF1-related GIST cells cultured in medium supplemented with stem cell factor (SCF) and control human fibroblasts determined by western blot assay. Equal protein loading was demonstrated with anti-actin staining. The relative p-MAPK expression was evaluated on the basis of the densitometric quantification of autoradiographic signals. In contrast to control GIST882 cells with activating KIT mutation, the phosphorylation of wild-type KIT in primary NF1-related GIST cells under in vitro condition is ligand-dependent. Imatinib treatment resulted in only partial and dose-independent inhibition of MAPK posphorylation of NF1-GIST cells cultured under ligand stimulation.

 

View this table:
[in this window]
[in a new window]
  		Table 4. Relative p-KIT and p-MAPK expression based on densitometric quantification of autoradiographic signals obtained by western blotting (data corresponding to Fig. 5)
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIAL AND METHODS
 REFERENCES
 
Although activating mutations of KIT and PDGFRA are known to be the most frequent genetic events in the development of sporadic GISTs, the underlying pathogenic mechanism leading to GIST formation in the NF1 setting remains elusive. In this study, total screening of KIT and PDGFRA in seven GISTs from three NF1 patients did not reveal any activating mutation. Several sequence variants were detected: three silent mutations in PDGFRA (p.N204N, p.G313G and p.A603A), 1 silent mutation in KIT (p.K546K), one SNP in the 3'-UTR region of both KIT and PDGFRA and one missense mutation in exon 10 of PDGFRA (p.S478P) that was not predicted to have an impact on protein activity (Polyphen algorithm) and that is a frequent polymorphism (19/98 controls, 19%) in the examined population. All variants were also present in normal control tissue (white blood cells) of the respective patients. In conclusion, the alterations do not correspond to known mutation hot spots in sporadic GISTs, are not tumor specific and have not been demonstrated to result in activation of the tyrosine kinases. This finding is in line with published reports (27Go–31Go). Of the 113 NF1-related GISTs previously published, only eight showed KIT/PDGFRA alterations. Takazawa et al. (2005) (30Go) reported three KIT (p.L558L, p.P627L and p.I653T) and two PDGFRA (p.P589S and p.R822S) mutations in 34 NF1-related GISTs. None have been demonstrated to result in activation of the protein nor have they been previously reported in sporadic or familial GIST. Yantiss et al. (2005) (29Go) detected an identical point mutation in KIT exon 11 in three tumors from one patient. As control tissue was not analyzed, this patient may have had a germline KIT mutation. Taken together, these results indicate that KIT or PDGFRA mutations are not playing a role in the development of GISTs in NF1 patients.

In accordance with Knudson's two-hit hypothesis (32Go), somatic inactivation of the NF1 tumor suppressor gene leads to tumor formation in NF1 (33Go–36Go). In line with this concept, we detected somatic NF1 alterations in six of seven NF1-related GISTs (Table 1). Analysis of different cell types derived from tumor II of patient NF1-2 demonstrated that the somatic NF1 mutation was only present in tumoral cells cultured using specific conditions for ICCs (37Go). This is the first time that somatic NF1 inactivation is demonstrated in neoplastic GIST cells and it illustrates that the lack of functional neurofibromin, in the absence of KIT or PDGFRA mutations, can lead to GIST formation.

Neurofibromin functions as a negative regulator of the MAP-kinase pathway by catalyzing the hydrolysis of active Ras-GTP to an inactive Ras-GDP. In this light, inactivation of neurofibromin in NF1-related GISTs is the mechanism leading to tumor formation. To further elucidate this hypothesis, we evaluated the expression of KIT, neurofibromin and several downstream signaling molecules. Clear differences were observed between sporadic and NF1-related GISTs (Fig. 2). First, NF1-related GISTs demonstrated comparable total KIT expression but low levels of constitutive KIT autophosphorylation. Secondly, in contrast to sporadic tumors, NF1-related GISTs showed a dramatically reduced level of neurofibromin, confirming the inactivating effect of the NF1 somatic mutations. Thirdly, strong expression and activation of MAPK in NF1-related GISTs illustrated increased signaling through the MAP-kinase pathway when compared with sporadic GIST cells. Fourthly, the JAK-STAT3 and PI3K-AKT pathways were shown to be less active in NF1-related GISTs. These data suggest that activation of the MAP-kinase cascade is a common cause of GIST development in both NF1 and non-NF1 patients, although there are important differences in the mechanism of activation (inactivation of NF1 versus activation of KIT/PDGFRA).

Recurrent cytogenetic aberrations in sporadic GISTs include deletion of 14q, 22q, 1p, 11p and 9p and gain of 8p and 17q (reviewed in 5). These chromosomal changes can be viewed as secondary events acquired after the oncogenic mutations in the receptor tyrosine kinases and are associated with clinical progression. While deletion of chromosome arms 14q (38Go–43Go) and 22q (39Go,42Go–45Go) are frequent findings in sporadic GISTs and likely represent sites for tumor suppressor genes that play a role early in GIST formation, losses on chromosomes 1p, 9p and 11p are more significantly associated with malignancy (40Go,46Go–48Go). Gains on chromosomes 8q and 17q are associated with metastatic behavior (41Go,42Go,49Go). The array CGH experiments on NF1-related GISTs showed similar gains and losses of chromosome regions as seen in sporadic GISTs, i.e. losses of chromosomes 11, 14, 22 and 1p, in addition to several extra alterations (Table 3). These additional chromosomal abnormalities seem not to be recurrent in the different tested NF1-related GISTs. Remarkable is the array CGH profile of the very large tumor from patient NF1-3 where only the clones containing the NF1 gene are deleted while no other aberrations are observed (Fig. 3). Taken together, the genomic changes observed in the analyzed NF1-related GISTs were similar to those described in sporadic GISTs and did not indicate any change that could be associated particularly with NF1-related GISTs, except for the deletion of the NF1 gene region. It remains elusive why clinically symptomatic GISTs arise in only a minority of NF1 patients and why, in contrast to sporadic tumors, they tend to be multiple and predominantly located in the small intestine. The presence of multiple GISTs in NF1 might reflect a distinct rate-limiting step in oncogenesis compared with sporadic GIST. While a broad spectrum of inactivating genetic mechanisms might lead to suppression of the wild-type NF1 allele and hence GIST formation in NF1 patients, only a limited set of specific activating mutations in KIT/PDGFRA will result in sporadic GIST. Moreover, genotypes at modifying loci, not detectable by array CGH, might trigger the development, location and number of multiple GISTs in NF1 patients.

On the basis of the array CGH profiles of the three tumors from patient NF1-2 and tumors I and II from patient NF1-1 (Table 3), one might argue that clustered GISTs are clonal. In this light, the identical chromosomal alterations in the tumors of each patient (loss 1pter->1p12 and 14 for NF1-2; loss 1pter->1p12, 14 and 22 for NF1-1) would reflect a common precursor lesion, whereas the tumor-specific alterations (cytogenetic aberrations and NF1 somatic mutation) could be seen as secondary events. However, as illustrated by the same alterations in the tumors from two different patients, deletion of chromosomes 1p, 14 and 22 are recurrent cytogenetic aberrations in GISTs. Moreover, the apparently identical breakpoints at chromosome band 1p12 can be explained by the poor clone coverage at the centromere [21 Mb gap between RP11-418J17 (1p12) and RP11-417J8 (1q12)]. Overall, different tumors derived from the same patient bear different NF1 somatic mutations and, with the exception of the typical GIST-related chromosomal alterations, array CGH profiles are quite diverse. Therefore, it is most likely that multiple GISTs in NF1 patients are independent tumors.

The tyrosine kinase inhibitor imatinib (Gleevec, Novartis) has tremendously improved the treatment of advanced sporadic GISTs, showing a clinical benefit in up to 80% of patients (17Go,18Go). To determine whether this drug could also be effective in NF1-related GISTs, we performed an ex vivo imatinib response experiment. As illustrated in Figure 5, ligand-dependent KIT autophosphorylation of primary NF1-related GIST cells and the control GIST882 cell line is totally inhibited by the exposure to imatinib at a concentration of 0.5 µM. In contrast to control fibroblasts, the level of MAPK autophosphorylation of primary NF1-related GIST cells was lowered after exposure to imatinib, although the inhibition was neither complete nor dose-dependent as clearly illustrated in the GIST882 cell line. These experiments thus suggest that imatinib might only have a mild effect on NF1-related GISTs. It will be important to determine whether this ex vivo observation can be translated into clinical benefit for NF1 patients with GISTs. As it was proven by in vitro studies (18Go), wild-type KIT is equally sensitive to imatinib as mutated KIT protein. Nevertheless, this observation does not translate to clinical response of patients with tumors that do not harbor KIT mutations (who poorly respond to the treatment). Therefore, in vitro assays may have only restricted predictive value.

In conclusion, we report for the first time the molecular pathogenesis of GISTs in NF1 individuals. Somatic NF1 inactivation in the ICCs seems to be the molecular event underlying GIST development in the NF1 setting. Inactivation of neurofibromin in the absence of KIT or PDGFRA mutations is an alternate mechanism leading to GIST formation. Moreover, NF1-related GISTs show an increased signaling through the MAP-kinase pathway when compared with sporadic GIST cell lines. These findings clearly position GISTs in the range of clinical symptoms seen in NF1. It will be important to determine whether the tyrosine kinase inhibitor Gleevec used in the treatment of sporadic GIST also has an effect on NF1-related GIST.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
REFERENCES
 
Patient material
Studies were performed on seven GISTs derived from three consenting NF1 patients. Peripheral blood was available for all patients as well as frozen tissue from the seven tumors and two tumor cell cultures from patient NF1-2. Control GIST cells included a GIST882 cell line harboring a KIT p.K642E mutation (50Go) and a sporadic GIST with a KIT p.557–558delWK mutation.

Phenotype NF1 patients
Patient NF1-1 is a 72-year-old female patient with widespread cutaneous neurofibromas. At the age of 59 years, a carcinoid tumor originating from the duodenum was resected. A GIST in the small intestine was diagnosed at the age of 68 and another in the retroperitoneal area adjacent to the bladder 4 years later. During surgery, both tumors were resected as well as a third not previously noted. The patient died post-operatively due to pulmonary complications. Her mother had had NF1 and had died due to complications related to a GIST at the age of 40.

Patient NF1-2 is a 59-year-old male with family history of neurofibromatosis type 1 and multiple cutaneous neurofibromas. Multiple GISTs of the duodenum were diagnosed by endoscopy following massive upper gastrointestinal tract bleeding. The episode of bleeding was preceded by the ingestion of a non-steroidal anti-inflammatory drug (nimesulide). During surgery, tumors were palpated at the serosal side of the duodenum and the proximal jejunum. No tumors could be palpated in the rest of the intestinal tract. Twelve small tumors (diameters of 3–22 mm) were removed from the duodenum and the proximal jejunum. A large lesion was localized in D2 and protruded at the anti-mesenterial side (diameter 20 mm). Two other large lesions (diameters 20 and 22 mm) showed mucosal ulcerations and were located in D2–D3. Pathological examination showed very few mitoses (less than 5 per 50 high-power fields). There were no post-operative complications, and post-operative follow-up at 2 years was uneventful. He did not receive adjuvant therapy.

Patient NF1-3 is a male with a severe thoraco-lumbar kyphoscoliosis and multiple cutaneous neurofibromas. At the age of 55, surgery was performed for a growing pelvic mass with nycturia and frequent micturition. The large tumor fills the pelvic cavity completely and is connected to the ileum over a distance of 4 cm. The large tumor was removed from the peritoneum, the bladder and the mesenterial side of the rectum and subsequently resected together with 7 cm of jejunum. The tumor measured 110x110x50 mm3 and showed central necrosis. Pathological examination showed only one mitosis per 50 high-power fields. In the resected fragment of the jejunum, a tumor with a diameter of 20 mm was present (no mitoses detected) and three additional smaller GISTs (diameter 3–5 mm, no mitoses). The post-operative evolution was uneventful and the patient did not receive adjuvant therapy. At follow-up 2.5 years later, he is free of symptoms and a CT-scan of the abdomen did not reveal any mass.

Histopathology
Histopathological sections (5 µm) were cut from tumor paraffin blocks for routine H&E staining. Immunohistochemical staining was conducted using the avidin–biotin–peroxidase complex method. Commercially available monoclonal (mc) or polyclonal (pc) antibodies specific for CD117 (pc, 1:250; DAKO, Glostrup, Denmark), CD34 (mc, 1:10; Becton Dickinson, San Jose, CA, USA), {alpha}-Smooth Muscle Actin (mc, 1:100; DAKO), desmin (mc, 1:20; ICN, Aurora, OH, USA) and S-100 (pc, 1:300; DAKO) were used. The CD117 immunostaining was performed without antigen retrieval and the presence of mast cells served as an internal control.

KIT/PDGFRA mutation analysis
Genomic DNA was extracted from snap-frozen tumor tissue of NF1-related GISTs using the High Pure PCR Template Preparation Kit (Roche). The entire KIT and PDGFRA coding region and the splice junctions were amplified by PCR. All amplicons were bidirectionally cycle sequenced using the ABI3730XL machine (Applied Biosystems).

NF1 mutation analysis
Germline NF1 mutation analysis was performed (51Go). An improved strategy for NF1 somatic mutation detection was developed (Maertens et al., manuscript in preparation) and applied specifically to the primary GIST cell cultures. Briefly, the second passage of ICC cell cultures from tumors II and III from patient NF1-2 was treated with puromycin before RNA extraction (RNeasy kit, Qiagen). Genomic DNA was extracted with the PureGene DNA purification kit (Gentra). The entire NF1 cDNA was sequenced. Mutations found at the cDNA level were confirmed on gDNA. Detection of LOH was performed by genotyping microsatellite markers telomeric (3'NF1-3, 3'NF1-1) (52Go) and within (Alu, IVS27AC33.1, IVS38GT53.0, IVS27TG24.8) (53Go–56Go) NF1. Genomic DNA from paired frozen tissue or primary GIST cell cultures and lymphocyte cultures was investigated by multiplex PCR and analyzed on the ABI3100 genetic analyzer (Applied Biosystems) with the Genescan software (Applied Biosystems). Loss of the wild-type NF1 allele was confirmed by at least one of the following techniques: FISH, array CGH, MLPA or SNP analysis. Frozen tumor specimens negative for LOH were subsequently submitted to PCR amplification and cycle sequencing of all NF1 exons.

Western blotting
Proteins were isolated from snap-frozen tumor specimens and western blotting was performed as described in Debiec-Rychter et al. (57Go). In short, equivalent amounts of protein (30 µg) from clarified lysates were resolved with SDS–PAGE, transferred to PDVF membranes and immunoblotted sequentially with antisera specific for anti-NF1 (sc-67; 1:200; Biotechnology, Santa Cruz, CA, USA), anti-phospho-KIT (Y703) (1:250; Zymed, San Francisco, CA, USA), anti-KIT (1:500; DAKO), anti-phospho MAPK (T202/Y204) (1:1000; Cell Signaling Technology Inc., Beverly, MA, USA), anti-MAPK (1:1000; Zymed), anti-phospho AKT (S473) (1:500; Cell Signaling Technology Inc.), anti-AKT (1:500; Cell Signaling Technology Inc.), anti-STAT3 (1:500; Zymed) and anti-actin (1:500; Sigma, Saint Louis, MI, USA). After washes, membranes were probed with anti-rabbit or anti-goat immunoglobulin-HRP conjugate and incubated with ECL substrate (Pierce, Rockford, IL, USA). On the basis of the densitometric quantification of autoradiographic signals, the relative p-MAPK expression was determined in NF1-related GISTs.

Array CGH
Array CGH was performed as published previously (58Go). Tumor DNA was compared with DNA extracted from blood lymphocytes of a sex-matched control individual. Every case was analyzed twice and tumor DNA was once labeled with Cy3 and once with Cy5. Clones that gave the same result twice were taken into account.

Primary GIST cell culture and imatinib assay
The GIST specimen used for this experiment was tumor III of patient NF1-2. Primary cells were obtained from a disaggregated tumor specimen seeded at 60–70% confluence in 100 mm cell culture dishes and grown for 3 days in DMEM supplemented with 10% FBS, 20 ng/ml SCF, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma). As a control, the same NF1-2 primary GIST cells, GIST882 cell line and normal human fibroblasts were grown in similar conditions but without the SCF ligand. For evaluation of the phenotype, NF1-2 primary GIST cells from tumor III were seeded at 60–70% confluence in slide chambers and grown for 3 days in the same conditions as above. After fixation in 4% paraformaldehyde, immunohistochemical staining using the polyclonal rabbit antihuman antibody against CD117 (1:250; DAKO) and Ki-67 antibody (1:250; DAKO) was performed by the avidin–biotin–peroxidase complex method. The proliferation index was determined as the percentage of Ki-67 positive cells of the total number of cells. Imatinib mesylate was provided by Novartis Pharmaceuticals Corporation. The compound was dissolved at 10 mM in 100% DMSO (Sigma). Controls were performed with solvent (DMSO) dilutions. The effect of imatinib on the autophosphorylation of the KIT Y703 and the MAPK T204/Y202 residues in cultured primary NF1-related GIST cells (either ligand stimulated or unstimulated), the control GIST882 cell line and human fibroblasts was determined by western blot assay. In short, cells were exposed to either vehicle alone or to imatinib mesylate within a range of doses (0.1, 0.5 and 1.0 µM) for 90 min, washed with ice-cold PBS and lysed. Lysates were incubated for 30 min at +4°C and then centrifuged for 20 min. Supernatants were removed and used for SDS gel electrophoresis and immunoblotting as described previously (57Go). Equivalency of loading intracellular proteins was demonstrated by stripping the membrane and re-blotting using anti-actin antibody (1:500; Sigma).


   ACKNOWLEDGEMENT
 
The authors thank Dr Thomy de Ravel for critically reading the manuscript. This work is supported by an Interuniversitary Attraction Poles grant from the Federal Office for Scientific, Technical and Cultural Affairs, Belgium (2002–2006; P5/25), by a Concerted Action Grant from the KULeuven and by a Concerted Action Grant from the UGent. E.L. is part-time clinical researcher of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO). This work is also supported by the FWO Vlaanderen (G.0096.02 to E.L. and G.0286.05 to M.D.-R.) and the Belgische Federatie tegen Kanker (SCIE2003-33 to E.L.). H.P. is supported by a grant from the Emmanuel Van der Schueren Foundation from the Vlaamse Liga tegen Kanker. A.W. benefits from a fellowship granted from the Federal Science Policy Office in order to promote the S&T cooperation with Central and Eastern Europe.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 

  1. Fletcher, C.D., Berman, J.J., Corless, C., Gorstein, F., Lasota, J., Longley, B.J., Miettinen, M., O'Leary, T.J., Remotti, H., Rubin, B.P. et al. (2002) Diagnosis of gastrointestinal stromal tumors: a consensus approach. Hum. Pathol., 33, 459–465.[CrossRef][ISI][Medline]

  2. Kindblom, L.G., Remotti, H.E., Aldenborg, F. and Meis-Kindblom, J.M. (1998) Gastrointestinal pacemaker cell tumor (GIPACT): gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal. Am. J. Pathol., 152, 1259–1269.[Abstract]

  3. Sircar, K., Hewlett, B.R., Huizinga, J.D., Chorneyko, K., Berezin, I. and Riddell, R.H. (1999) Interstitial cells of Cajal as precursors of gastrointestinal stromal tumors. Am. J. Surg. Pathol., 23, 377–389.[CrossRef][ISI][Medline]

  4. Hirota, S., Isozaki, K., Moriyama, Y., Hashimoto, K., Nishida, T., Ishiguro, S., Kawano, K., Hanada, M., Kurata, A., Takeda, M. et al. (1998) Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science, 279, 577–580.[Abstract/Free Full Text]

  5. Corless, C.L., Fletcher, J.A. and Heinrich, M.C. (2004) Biology of gastrointestinal stromal tumors. J. Clin. Oncol., 22, 3813–3825.[Abstract/Free Full Text]

  6. Rubin, B.P., Singer, S., Tsao, C., Duensing, A., Lux, M.L., Ruiz, R., Hibbard, M.K., Chen, C.J., Xiao, S., Tuveson, D.A. et al. (2001) KIT activation is a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res., 61, 8118–8121.[Abstract/Free Full Text]

  7. Heinrich, M.C., Corless, C.L., Duensing, A., McGreevey, L., Chen, C.J., Joseph, N., Singer, S., Griffith, D.J., Haley, A., Town, A. et al. (2003) PDGFRA activating mutations in gastrointestinal stromal tumors. Science, 299, 708–710.[Abstract/Free Full Text]

  8. Hirota, S., Ohashi, A., Nishida, T., Isozaki, K., Kinoshita, K., Shinomura, Y. and Kitamura, Y. (2003) Gain-of-function mutations of platelet-derived growth factor receptor alpha gene in gastrointestinal stromal tumors. Gastroenterology, 125, 660–667.[CrossRef][ISI][Medline]

  9. Schlessinger, J. (2000) Cell signaling by receptor tyrosine kinases. Cell, 103, 211–225.[CrossRef][ISI][Medline]

  10. Porfiri, E. and McCormick, F. (1996) Regulation of epidermal growth factor receptor signaling by phosphorylation of the ras exchange factor hSOS1. J. Biol. Chem., 271, 5871–5877.[Abstract/Free Full Text]

  11. Burack, W.R. and Sturgill, T.W. (1997) The activating dual phosphorylation of MAPK by MEK is nonprocessive. Biochemistry, 36, 5929–5933.[CrossRef][Medline]

  12. Ihle, J.N. and Kerr, I.M. (1995) Jaks and Stats in signaling by the cytokine receptor superfamily. Trends Genet., 11, 69–74.[CrossRef][ISI][Medline]

  13. Schindler, C. and Darnell, J.E., Jr. (1995) Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu. Rev. Biochem., 64, 621–651.[ISI][Medline]

  14. Weiler, S.R., Mou, S., DeBerry, C.S., Keller, J.R., Ruscetti, F.W., Ferris, D.K., Longo, D.L. and Linnekin, D. (1996) JAK2 is associated with the c-kit proto-oncogene product and is phosphorylated in response to stem cell factor. Blood, 87, 3688–3693.[Abstract/Free Full Text]

  15. Brizzi, M.F., Dentelli, P., Rosso, A., Yarden, Y. and Pegoraro, L. (1999) STAT protein recruitment and activation in c-Kit deletion mutants. J. Biol. Chem., 274, 16965–16972.[Abstract/Free Full Text]

  16. Mui, A.L. (1999) The role of STATs in proliferation, differentiation, and apoptosis. Cell. Mol. Life Sci., 55, 1547–1558.[CrossRef][ISI][Medline]

  17. van Oosterom, A.T., Judson, I., Verweij, J., Stroobants, S., Donato di Paola, E., Dimitrijevic, S., Martens, M., Webb, A., Sciot, R., Van Glabbeke, M. et al. (2001) Safety and efficacy of imatinib (STI571) in metastatic gastrointestinal stromal tumours: a phase I study. Lancet, 358, 1421–1423.[CrossRef][ISI][Medline]

  18. Heinrich, M.C., Corless, C.L., Demetri, G.D., Blanke, C.D., von Mehren, M., Joensuu, H., McGreevey, L.S., Chen, C.J., Van den Abbeele, A.D., Druker, B.J. et al. (2003) Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J. Clin. Oncol., 21, 4342–4349.[Abstract/Free Full Text]

  19. Xu, G.F., Lin, B., Tanaka, K., Dunn, D., Wood, D., Gesteland, R., White, R., Weiss, R. and Tamanoi, F. (1990) The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell, 63, 835–841.[CrossRef][ISI][Medline]

  20. Wallace, M.R., Marchuk, D.A., Andersen, L.B., Letcher, R., Odeh, H.M., Saulino, A.M., Fountain, J.W., Brereton, A., Nicholson, J., Mitchell, A.L. et al. (1990) Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients. Science, 249, 181–186.[Abstract/Free Full Text]

  21. Viskochil, D., Buchberg, A.M., Xu, G., Cawthon, R.M., Stevens, J., Wolff, R.K., Culver, M., Carey, J.C., Copeland, N.G., Jenkins, N.A. et al. (1990) Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell, 62, 187–192.[CrossRef][ISI][Medline]

  22. Martin, G.A., Viskochil, D., Bollag, G., McCabe, P.C., Crosier, W.J., Haubruck, H., Conroy, L., Clark, R., O'Connell, P., Cawthon, R.M. et al. (1990) The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell, 63, 843–849.[CrossRef][ISI][Medline]

  23. Ballester, R., Marchuk, D., Boguski, M., Saulino, A., Letcher, R., Wigler, M. and Collins, F. (1990) The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell, 63, 851–859.[CrossRef][ISI][Medline]

  24. Riccardi, V.M. (1992) Neurofibromatosis: Phenotype, Natural History and Pathogenesis. The John Hopkins University Press, Baltimore and London.

  25. Zoller, M.E., Rembeck, B., Oden, A., Samuelsson, M. and Angervall, L. (1997) Malignant and benign tumors in patients with neurofibromatosis type 1 in a defined Swedish population. Cancer, 79, 2125–2131.[CrossRef][ISI][Medline]

  26. Miettinen, M., Kopczynski, J., Makhlouf, H.R., Sarlomo-Rikala, M., Gyorffy, H., Burke, A., Sobin, L.H. and Lasota, J. (2003) Gastrointestinal stromal tumors, intramural leiomyomas, and leiomyosarcomas in the duodenum: a clinicopathologic, immunohistochemical, and molecular genetic study of 167 cases. Am. J. Surg. Pathol., 27, 625–641.[CrossRef][ISI][Medline]

  27. Miettinen, M., Fetsch, J.F., Sobin, L.H. and Lasota, J. (2006) Gastrointestinal stromal tumors in patients with neurofibromatosis 1: a clinicopathologic and molecular genetic study of 45 cases. Am. J. Surg. Pathol., 30, 90–96.[CrossRef][ISI][Medline]

  28. Andersson, J., Sihto, H., Meis-Kindblom, J.M., Joensuu, H., Nupponen, N. and Kindblom, L.G. (2005) NF1-associated gastrointestinal stromal tumors have unique clinical, phenotypic, and genotypic characteristics. Am. J. Surg. Pathol., 29, 1170–1176.[CrossRef][ISI][Medline]

  29. Yantiss, R.K., Rosenberg, A.E., Sarran, L., Besmer, P. and Antonescu, C.R. (2005) Multiple gastrointestinal stromal tumors in type I neurofibromatosis: a pathologic and molecular study. Mod. Pathol., 18, 475–484.[CrossRef][ISI][Medline]

  30. Takazawa, Y., Sakurai, S., Sakuma, Y., Ikeda, T., Yamaguchi, J., Hashizume, Y., Yokoyama, S., Motegi, A. and Fukayama, M. (2005) Gastrointestinal stromal tumors of neurofibromatosis type I (von Recklinghausen's disease). Am. J. Surg. Pathol., 29, 755–763.[CrossRef][ISI][Medline]

  31. Kinoshita, K., Hirota, S., Isozaki, K., Ohashi, A., Nishida, T., Kitamura, Y., Shinomura, Y. and Matsuzawa, Y. (2004) Absence of c-kit gene mutations in gastrointestinal stromal tumours from neurofibromatosis type 1 patients. J. Pathol., 202, 80–85.[CrossRef][ISI][Medline]

  32. Knudson, A.G., Jr. (1971) Mutation and cancer: statistical study of retinoblastoma. Proc. Natl Acad. Sci. USA, 68, 820–823.[Abstract/Free Full Text]

  33. Xu, W., Mulligan, L.M., Ponder, M.A., Liu, L., Smith, B.A., Mathew, C.G. and Ponder, B.A. (1992) Loss of NF1 alleles in phaeochromocytomas from patients with type I neurofibromatosis. Genes Chromosomes Cancer, 4, 337–342.[ISI][Medline]

  34. Shannon, K.M., O'Connell, P., Martin, G.A., Paderanga, D., Olson, K., Dinndorf, P. and McCormick, F. (1994) Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders. N. Engl. J. Med., 330, 597–601.[Abstract/Free Full Text]

  35. Legius, E., Marchuk, D.A., Collins, F.S. and Glover, T.W. (1993) Somatic deletion of the neurofibromatosis type 1 gene in a neurofibrosarcoma supports a tumour suppressor gene hypothesis. Nat. Genet., 3, 122–126.[CrossRef][ISI][Medline]

  36. Colman, S.D., Williams, C.A. and Wallace, M.R. (1995) Benign neurofibromas in type 1 neurofibromatosis (NF1) show somatic deletions of the NF1 gene. Nat. Genet., 11, 90–92.[CrossRef][ISI][Medline]

  37. Li, C.X., Liu, B.H., Tong, W.D., Zhang, L.Y. and Jiang, Y.P. (2005) Dissociation, culture and morphologic changes of interstitial cells of Cajal in vitro. World J. Gastroenterol., 11, 2838–2840.[Medline]

  38. Heinrich, M.C., Rubin, B.P., Longley, B.J. and Fletcher, J.A. (2002) Biology and genetic aspects of gastrointestinal stromal tumors: KIT activation and cytogenetic alterations. Hum. Pathol., 33, 484–495.[CrossRef][ISI][Medline]

  39. Fukasawa, T., Chong, J.M., Sakurai, S., Koshiishi, N., Ikeno, R., Tanaka, A., Matsumoto, Y., Hayashi, Y., Koike, M. and Fukayama, M. (2000) Allelic loss of 14q and 22q, NF2 mutation, and genetic instability occur independently of c-kit mutation in gastrointestinal stromal tumor. Jpn. J. Cancer Res., 91, 1241–1249.[CrossRef][ISI][Medline]

  40. El-Rifai, W., Sarlomo-Rikala, M., Miettinen, M., Knuutila, S. and Andersson, L.C. (1996) DNA copy number losses in chromosome 14: an early change in gastrointestinal stromal tumors. Cancer Res., 56, 3230–3233.[Abstract/Free Full Text]

  41. El-Rifai, W., Sarlomo-Rikala, M., Andersson, L.C., Miettinen, M. and Knuutila, S. (2000) High-resolution deletion mapping of chromosome 14 in stromal tumors of the gastrointestinal tract suggests two distinct tumor suppressor loci. Genes Chromosomes Cancer, 27, 387–391.[CrossRef][ISI][Medline]

  42. Debiec-Rychter, M., Lasota, J., Sarlomo-Rikala, M., Kordek, R. and Miettinen, M. (2001) Chromosomal aberrations in malignant gastrointestinal stromal tumors: correlation with c-KIT gene mutation. Cancer Genet. Cytogenet., 128, 24–30.[CrossRef][ISI][Medline]

  43. Chen, Y., Tzeng, C.C., Liou, C.P., Chang, M.Y., Li, C.F. and Lin, C.N. (2004) Biological significance of chromosomal imbalance aberrations in gastrointestinal stromal tumors. J. Biomed. Sci., 11, 65–71.[CrossRef][ISI][Medline]

  44. Pylkkanen, L., Sarlomo-Rikala, M., Wessman, M., Hamalainen, E., Sainio, M., Husgafvel-Pursiainen, K. and Carpen, O. (2003) Chromosome 22q alterations and expression of the NF2 gene product, merlin, in gastrointestinal stromal tumors. Hum. Pathol., 34, 872–879.[CrossRef][ISI][Medline]

  45. Lasota, J., Wozniak, A., Kopczynski, J., Dansonka-Mieszkowska, A., Wasag, B., Mitsuhashi, T., Sarlomo-Rikala, M., Lee, J.R., Schneider-Stock, R., Stachura, J. et al. (2005) Loss of heterozygosity on chromosome 22q in gastrointestinal stromal tumors (GISTs): a study on 50 cases. Lab. Invest., 85, 237–247.[CrossRef][ISI][Medline]

  46. O'Leary, T., Ernst, S., Przygodzki, R., Emory, T. and Sobin, L. (1999) Loss of heterozygosity at 1p36 predicts poor prognosis in gastrointestinal stromal/smooth muscle tumors. Lab. Invest., 79, 1461–1467.[ISI][Medline]

  47. Perrone, F., Tamborini, E., Dagrada, G.P., Colombo, F., Bonadiman, L., Albertini, V., Lagonigro, M.S., Gabanti, E., Caramuta, S., Greco, A. et al. (2005) 9p21 locus analysis in high-risk gastrointestinal stromal tumors characterized for c-kit and platelet-derived growth factor receptor alpha gene alterations. Cancer, 104, 159–169.[CrossRef][ISI][Medline]

  48. Sabah, M., Cummins, R., Leader, M. and Kay, E. (2004) Loss of heterozygosity of chromosome 9p and loss of p16INK4A expression are associated with malignant gastrointestinal stromal tumors. Mod. Pathol., 17, 1364–1371.[CrossRef][ISI][Medline]

  49. El-Rifai, W., Sarlomo-Rikala, M., Andersson, L.C., Knuutila, S. and Miettinen, M. (2000) DNA sequence copy number changes in gastrointestinal stromal tumors: tumor progression and prognostic significance. Cancer Res., 60, 3899–3903.[Abstract/Free Full Text]

  50. Tuveson, D.A., Willis, N.A., Jacks, T., Griffin, J.D., Singer, S., Fletcher, C.D., Fletcher, J.A. and Demetri, G.D. (2001) STI571 inactivation of the gastrointestinal stromal tumor c-KIT oncoprotein: biological and clinical implications. Oncogene, 20, 5054–5058.[CrossRef][ISI][Medline]

  51. Messiaen, L.M., Callens, T., Mortier, G., Beysen, D., Vandenbroucke, I., Van Roy, N., Speleman, F. and Paepe, A.D. (2000) Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum. Mutat., 15, 541–555.[CrossRef][ISI][Medline]

  52. Lopez Correa, C., Brems, H., Lazaro, C., Estivill, X., Clementi, M., Mason, S., Rutkowski, J.L., Marynen, P. and Legius, E. (1999) Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions. Hum. Mutat., 14, 387–393.[CrossRef][ISI][Medline]

  53. Xu, G.F., Nelson, L., O'Connell, P. and White, R. (1991) An Alu polymorphism intragenic to the neurofibromatosis type 1 gene (NF1). Nucleic Acids Res., 19, 3764.[Free Full Text]

  54. Lazaro, C., Gaona, A., Xu, G., Weiss, R. and Estivill, X. (1993) A highly informative CA/GT repeat polymorphism in intron 38 of the human neurofibromatosis type 1 (NF1) gene. Hum. Genet., 92, 429–430.[CrossRef][ISI][Medline]

  55. Lazaro, C., Gaona, A., Ravella, A., Volpini, V., Casals, T., Fuentes, J.J. and Estivill, X. (1993) Novel alleles, hemizygosity and deletions at an Alu-repeat within the neurofibromatosis type 1 (NF1) gene. Hum. Mol. Genet., 2, 725–730.[Abstract/Free Full Text]

  56. Lazaro, C., Gaona, A. and Estivill, X. (1994) Two CA/GT repeat polymorphisms in intron 27 of the human neurofibromatosis (NF1) gene. Hum. Genet., 93, 351–352.[CrossRef][ISI][Medline]

  57. Debiec-Rychter, M., Wasag, B., Stul, M., De Wever, I., Van Oosterom, A., Hagemeijer, A. and Sciot, R. (2004) Gastrointestinal stromal tumours (GISTs) negative for KIT (CD117 antigen) immunoreactivity. J. Pathol., 202, 430–438.[CrossRef][ISI][Medline]

  58. Vermeesch, J.R., Melotte, C., Froyen, G., Van Vooren, S., Dutta, B., Maas, N., Vermeulen, S., Menten, B., Speleman, F., De Moor, B. et al. (2005) Molecular karyotyping: array CGH quality criteria for constitutional genetic diagnosis. J. Histochem. Cytochem., 53, 413–422.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Roentgenol.Home page
M. A. Bredella, M. Torriani, F. Hornicek, H. A. Ouellette, W. E. Plamer, Z. Williams, A. J. Fischman, and S. R. Plotkin
Value of PET in the Assessment of Patients with Neurofibromatosis Type 1
Am. J. Roentgenol., October 1, 2007; 189(4): 928 - 935.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
B. Pasini, L. Matyakhina, T. Bei, M. Muchow, S. Boikos, B. Ferrando, J. A. Carney, and C. A. Stratakis
Multiple Gastrointestinal Stromal and Other Tumors Caused by Platelet-Derived Growth Factor Receptor {alpha} Gene Mutations: A Case Associated with a Germline V561D Defect
J. Clin. Endocrinol. Metab., September 1, 2007; 92(9): 3728 - 3732.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
D. R Stewart, C. L Corless, B. P Rubin, M. C Heinrich, L. M Messiaen, L. J Kessler, P. J Zhang, and D. G Brooks
Mitotic recombination as evidence of alternative pathogenesis of gastrointestinal stromal tumours in neurofibromatosis type 1
J. Med. Genet., January 1, 2007; 44(1): e61 - e61.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available