Volume 131, Issue 6, Pages 1907-1912 (December 2006) Intestinal Neurofibromatosis Is a Subtype of Familial GIST and Results From a Dominant Activating Mutation in PDGFRA  Thomas de Raedt, Jan Cools, Maria Debiec–Rychter, Hilde Brems, Nicole Mentens, Raf Sciot, Jacques Himpens, Ivo de Wever, Patrick Schöffski, Peter Marynen, Eric Legius  Gastroenterology  Volume 131, Issue 6, Pages 1907-1912 (December 2006) DOI: 10.1053/j.gastro.2006.07.002 Copyright © 2006 AGA Institute Terms and Conditions

Figure 1 (A) Family tree: Features indicated are bowel obstruction, intestinal tumors, large hands, and coarse face and the presence of a translocation. Individual II.3 died from bowel obstruction and had intestinal tumors; no data were available on the presence of the translocation or the large hands and coarse face. Individual I.2 died from bowel obstruction; all other features remain unknown. Individual III.1 is the proband investigated in Heimann et al.1 (B) The sequence shows the mutation and amino acid change in individual III.1. Gastroenterology 2006 131, 1907-1912DOI: (10.1053/j.gastro.2006.07.002) Copyright © 2006 AGA Institute Terms and Conditions

Figure 2 Immunostaining and in situ hybridization of tumors. (A) PDGFRA(Y555C) tumor: low-power view, showing the poorly cellular spindle cell proliferation in a loosely textured collagenous background. Note the wavy aspect of the collagen fibers and tumor cells. Arrows indicate mast cells. H&E stain; original magnification, 100×. (B) PDGFRA(Y555C) tumor: S100 stain: expression of S100 is only observed in the myenteric plexus of the outer muscular wall (left upper part), while the tumor is negative (right lower part). Indirect immunoperoxidase; original magnification, 50×. (C) PDGFRA(Y555C) tumor: c-kit stain: The c-kit stain only reveals expression in mast cells and not in any of the other cells. Indirect immunoperoxidase; original magnification, 200×. (D) PDGFRA(Y555C) tumor: detail of the negative S100 stain. Indirect immunoperoxidase, original magnification, 200×. (E) PDGFRA(Y555C) tumor: in situ hybridization of DOG1, no expression of DOG1; original magnification, 100×. (F) PDGFRA(D842V) control tumor: in situ hybridization of DOG1, high expression of DOG1; original magnification, 100×. Gastroenterology 2006 131, 1907-1912DOI: (10.1053/j.gastro.2006.07.002) Copyright © 2006 AGA Institute Terms and Conditions

Figure 3 PDGFRα(Y555C) is activated in intestinal tumors. Western blot analysis of the level of activation of PDGFRα, and the expression of KIT and PKC-θ in 2 intestinal tumors (lanes 2 and 3) and skin fibroblasts (lane 4) from patient III.3, and in a control sporadic PDGFRα(D842V) (lane 1), KIT(K642E) (lane 5), and KIT(V559D) (lane 6) mutant GISTs are shown.18 Sporadic GISTs are indicated by asterisk. Intestinal tumors from patient III.3 show a clear but relatively weak PDGFRα level of activation (phosphorylation) in comparison with sporadic PDGFRα(D842V) mutant GIST, whereas the patient III.3 skin fibroblasts and sporadic GIST KIT mutants do not show expression of PDGFRα. The KIT and PKC-θ is expressed only in sporadic PDGFRα or KIT mutant GISTs (kDa, kilodalton). Gastroenterology 2006 131, 1907-1912DOI: (10.1053/j.gastro.2006.07.002) Copyright © 2006 AGA Institute Terms and Conditions

Figure 4 PDGFRα(Y555C) is an activated tyrosine kinase that is sensitive to imatinib inhibition. (A) PDGFRα(Y555C), PDGFRα wild type, and PDGFRα(D842V) were expressed in 293T cells in increasing concentrations, by transfecting different amounts of the indicated constructs (total amount of DNA transfected was kept constant). No ligand was added to the cultures. The Y555C and D842V mutants show a similar pattern of activation, whereas wild-type PDGFRα is only weakly activated at the highest protein expression level. (B) PDGFRα(Y555C) was not activated when expressed in Ba/F3 cells, whereas D842V was activated in this cell line. PDGFRα(Y555C) could be activated by addition of PDGF-AA ligand. (C) Inhibition of PDGFRα(Y555C) by imatinib. The Y555C and D842V mutants were expressed in 293T cells, and the cells were treated with different concentrations of imatinib. The Y555C mutant was clearly inhibited by imatinib, as illustrated by the absence of autophosphorylation at 0.5 μmol/L and 1.0 μmol/L imatinib, whereas the D842V mutant was resistant to imatinib (as shown previously). The presence of the higher molecular weight is caused by the accumulation of mature PDGFRα at the plasma membrane after treatment with imatinib. Gastroenterology 2006 131, 1907-1912DOI: (10.1053/j.gastro.2006.07.002) Copyright © 2006 AGA Institute Terms and Conditions