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Keywords:

  • gastrointestinal stromal tumor;
  • insulin like growth factor 1 receptor;
  • pediatric

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Wildtype (WT) gastrointestinal stromal tumors (GISTs), lacking mutations in KIT or PDGFRA, represent 85% of GISTs in pediatric patients. Treatment options for pediatric WT GIST are limited. Recently, expression profiling of a limited number of pediatric and adult WT GISTs and more in depth study of a single pediatric WT GIST implicated the insulin-like growth factor 1 receptor (IGF1R) as a potential therapeutic target in pediatric WT GIST. We performed immunoblotting, SNP and FISH studies to determine the extent of expression, biochemical activation and genomic amplification of IGF1R in a larger number of pediatric WT GISTs. Pediatric WT GISTs expressed IGF1R strongly, whereas typical adult KIT mutant GISTs did not. IGF1R gene amplification was not detected in pediatric WT GISTs, and some KIT-mutant GISTs had IGF1R gene deletion due to monosomy 15. Despite the absence of apparent genomic activation mechanisms accounting for overexpression, clinical study of IGF1R-directed therapies in pediatric WT GIST is warranted.

The insulin-like growth factor 1 receptor (IGF1R) and its ligands, insulin-like growth factors (IGF) 1 and 2 serve crucial physiologic roles in growth and development.1, 2 The IGF pathway also has important pathophysiologic roles in cancer: IGF1R is required for neoplastic transformation in many experimental systems3–5; IGF1R is expressed strongly in a variety of neoplasms; and IGFs promote proliferation of neoplastic cells.6, 7 Monoclonal antibodies and small molecules targeting IGF1R are currently undergoing clinical evaluation. In early phase clinical trials of anti-IGF1R antibodies, adverse effects have been minimal and objective responses have been documented in advanced neuroendocrine tumors, Ewing sarcoma and osteosarcoma.8–10

Gastrointestinal stromal tumor (GIST), the most common mesenchymal neoplasm of the gastrointestinal tract, is resistant to conventional cytotoxic chemotherapy.11 Mutations in KIT or PDGFRA are present in 85% of GISTs occurring in adults. Imatinib and sunitinib, small-molecule inhibitors of the mutant KIT and PDGFRA receptor tyrosine kinases, significantly prolong survival in patients with GIST.12, 13 However, more than 85% of GISTs in children are wild-type (WT), lacking detectable mutations in KIT or PDGFRA.14 Imatinib appears to be less effective against these WT tumors than against GIST harboring activating mutations.15, 16 Sunitinib therapy only rarely results in objective responses in children and adults with WT GIST.16, 17

A recent study demonstrated strong IGF1R expression and low level IGF1R gene amplification in several adult WT GISTs and a single pediatric WT GIST. A subsequent study confirmed high IGF1R expression in 2 adult WT GISTs but did not find associated IGF1R gene amplification.18 These studies implicate IGF1R as a potential therapeutic target in adult WT GIST.19–21 However, results in adult WT GIST do not necessarily apply to pediatric WT GIST because in prior studies, pediatric and adult WT GISTs have been found to have distinct clinical and biological features.20, 22 Because additional preclinical data would help determine whether clinical trials of IGF1R-directed therapy in pediatric GIST are warranted, we evaluated a more sizeable sampling of pediatric GIST specimens for multiple characteristics, including IGF1R protein expression, IGF1R activation and IGF1R gene copy number.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Western blotting

The level of IGF1R expression in pediatric WT GISTs vs. adult KIT-mutant GISTs was analyzed by western blot. Whole cell lysates were prepared from 14 cryopreserved GISTs for which KIT and PDGFRA mutation status had already been determined.14 Lysates from 9 pediatric WT GISTs (cases P1-P9) and 5 KIT-mutant GISTs (cases M1-M5) were subjected to gel electrophoresis as previously described.14, 23 Blots were stained with antibodies to IGF1R (Cell signaling, Beverly, MA) and actin (Sigma, St. Louis, MO). Additional staining of the GIST immunoblots for activated IGF1R expression was performed using an antibody to phospho-IGF1R Y1135/1136 (Cell signaling, Beverly, MA).

SNP array and analysis

We evaluated IGF1R gene amplification in pediatric WT GIST using single nucleotide polymorphism (SNP) arrays. Genomic DNA isolated from 14 cryopreserved pediatric WT GISTs, 3 cryopreserved adult KIT-mutant GISTs and 4 normal control samples was digested with the StyI restriction enzyme. Digested DNA was then ligated to an adaptor before subsequent PCR amplification using AmpliTaq Gold (Applied Biosystems, Foster City, CA). PCR products were pooled, concentrated and fragmented with DNase I to a size range of 200–1,100 bp. Fragmented PCR products were then labeled, denatured and hybridized to Affymetrix 250K Sty SNP arrays interrogating ∼238,000 SNPs. After hybridization, the arrays were washed on the Affymetrix fluidics stations, stained and scanned using the Gene Chip Scanner 3000 7G and the genotyping software Affymetrix Genotyping Tools Version 2.0. DNA isolation and data analysis were performed as previously described.14, 24

IGF1R fluorescence in situ hybridization (FISH)

FISH was performed in 4-μm formalin-fixed paraffin-embedded tissue sections. Hybridization and signal detection techniques were as described previously.25 The IGF1R probe was composed of 2 overlapping BAC clones, RP11-262P8 and RP11-654A16, labeled by random priming with digoxigenin and detected with FITC antidigoxigenin, and cohybridized with a spectrum orange-labeled chromosome 15 pericentromeric probe (CEP15 D15Z4; Abbott Molecular). IGF1R to chromosome 15 centromeric ratio was determined in 100 nuclei.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

High-level IGF1R expression was detected in 8 of 9 pediatric WT GISTs, whereas all 5 KIT-mutant GISTs lacked IGF1R expression (Fig. 1). All pediatric WT GISTs and case M5, a KIT mutant GIST lacking IGF1R expression, appeared to express phosphorylated IGF1R (Fig. 1b). All of the pediatric WT GISTs featured AKT activation (Fig. 1b).

thumbnail image

Figure 1. Western blotting of IGF1R, KIT and AKT shows strong IGF1R expression in pediatric WT GISTs but not in comparison KIT-mutant GISTs.

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By SNP analysis, none of the pediatric WT GISTs had IGF1R gene amplification (Fig. 2a). The SNP results were validated by fluorescence in situ hybridization (FISH) in pediatric WT GISTs P5 and P7 (Fig. 2b). FISH confirmed the absence of IGF1R gene amplification in these 2 pediatric WT GISTs and in one additional pediatric WT GIST (P15) for which there was insufficient fresh frozen specimen for SNP analysis (Fig. 2c).

thumbnail image

Figure 2. Copy number at the IGF1R locus as determined by SNP array study (a) and FISH (b and c) of IGF1R (green) and chromosome 15 centromere (red) demonstrates lack of IGF1R gene amplification in pediatric WT GIST.

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The 3 KIT-mutant cases, M1, M2 and M3 included in the SNP study had decreased copy number at the IGF1R locus due to monosomy 15, which is a common cytogenetic finding in adult KIT-mutant GIST.26 Three additional adult KIT mutant cases were analyzed by FISH and had IGF1R to chromosome 15 centromeric ratios of 0.98 to 1.54. Notably, Case 5, which had the IGF1R to chromosome 15 centromeric ratio of 1.54 did not express IGF1R.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our immunoblotting results demonstrating high IGF1R expression in pediatric WT GISTs are in agreement with transcriptional profiling of 8 pediatric WT GISTs performed by Agaram20 and with immunohistochemistry of a single pediatric WT GIST performed by Tarn.19 The consistent high-level IGF1R expression in pediatric WT GISTs and the stark contrast with low-level IGF1R expression in KIT-mutant GISTs demonstrated in this study further implicates IGF1R as a potential therapeutic target in pediatric WT GIST. Similar to this study, Tarn et al. found a lack of correlation between phosphorylated IGF1R and total IGF1R expression.19 This might result from cross reactivity of the phospho-IGF1R antibody with the phophorylated insulin receptor, which has close homology to IGF1R. All of the pediatric WT GISTs studied featured AKT activation but we have previously reported KIT activation in these same tumors.14 Hence, it is unclear whether activation of downstream survival and growth pathways in pediatric WT GIST is IGF1R-dependent, KIT-dependent, or both. Therefore, these studies, although consistent with IGF1R activation, are not definitive.

In a prior study, low level IGF1R gene amplification was detected in WT GISTs in adults and in a single pediatric GIST.19 However, in a recent publication by Pantaleo et al.,18 2 WT GISTs had high IGF1R expression without IGF1R gene amplification. Our SNP array and FISH results demonstrate that the strong IGF1R expression present in pediatric WT GISTs is not due to gene amplification. The KIT-mutant GISTs we studied had low-to-absent IGF1R expression. In some of the cases, absent IGF1R expression could potentially be due to monosomy 15. However, absent IGF1R expression was also seen in a KIT-mutant GIST with 2 copies of IGF1R and so lack of IGF1R expression in adult KIT-mutant GIST cannot be attributed entirely to IGF1R gene deletion.

In conclusion, IGF1R is expressed strongly in pediatric WT GISTs but this is not due to detectable gene amplification. Further preclinical evaluation of the IGF1R pathway in pediatric GISTs such as drug-response studies in cell lines or animal models are warranted. Unfortunately, attempts to develop such models of pediatric and adult WT GIST have not yet been successful. These results support clinical evaluation of anti-IGF1R antibody therapies in pediatric patients with metastatic WT GIST especially given the relatively mild toxicity profile of anti-IGF1R antibodies and the limited alternative therapeutic options.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Yue Xiang Wang and Cher-Wei Liang for assistance with FISH.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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