Mutations in exon 11 of c-Kit occur preferentially in malignant versus benign gastrointestinal stromal tumors and do not occur in leiomyomas or leiomyosarcomas

Abstract

Gastrointestinal stromal tumors (GISTs) comprise the largest subset of mesenchymal tumors of the gastrointestinal tract. These neoplasms differ histologically and immunohistochemically from typical leiomyomas and leiomyosarcomas. Most GISTs express CD34 and CD117 (c-kit protein) but not desmin. Recently, gain-of-function mutations of c-kit proto-oncogene have been shown in five solitary GISTs and in tumors and leukocytes from a family with multiple GISTs. An in-frame deletion or a point mutation in exon 11 of c-kit was detected in these cases. Stable transfection of the mutant c-kit complementary DNA was also shown to induce malignant transformation of murine lymphoid cells, suggesting that the c-kit mutations contribute to tumor development. In this study, we evaluated 43 GISTs and 14 smooth muscle tumors for mutations in the exon 11 of c-kit by a PCR-assay. Half of the malignant GISTs (12/24) and only one benign GIST (1/19) revealed mutant bands. No mutant bands were found in 3 leiomyomas and 11 leiomyosarcomas. Sequence analysis confirmed the presence of an in-frame deletion of 3-21 bp in all 13 GISTs with mutant bands. Wild-type bands from 8 malignant and 11 benign GISTs and 7 smooth muscle tumors without mutant bands were cloned and sequenced. Additional mutations were found in 3 malignant and 2 benign GISTs. There were no mutations in 3 leiomyomas and 4 leiomyosarcomas. The mutation status of exon 11 did not correlate with immunohistochemically detectable expression of the CD117, as virtually all GISTs with or without such mutations showed CD117 immunoreactivity. The c-kit mutations occur preferentially in malignant GISTs and might be a clinically useful adjunct marker in the evaluation of GISTs. The conservation of the c-kit mutation pattern, observed in consecutive lesions from the same patients, suggests that these mutations might be useful tumor markers in monitoring recurrence or minimal residual disease.

Figure 1.

A benign small intestinal GIST of spindle cell type. Note the uniform appearance of bland spindle cells without mitotic activity. Hematoxylin and eosin, original magnification ×411.

Figure 2.

A benign spindle cell GIST (shown in Figure 1 ▶ ) is strongly positive for CD117 (left), but negative for α-smooth muscle actin; vessel walls are positive (right). ABC immunoperoxidase, original magnification ×300.

Figure 3.

Sequences of the exon 11 of c-kit from the mutant bands obtained from the GISTs. The deleted nucleotides are indicated with x, and point mutations with ▪. The fragments of germline sequence of exon 11 are shown according to Andre et al. The codons are indicated above with numbers and the gray area indicates codons 563–566.

Figure 4.

Predicted amino acid sequences of the mutant c-kit. The sequence starts at codon 550 and ends at 580. The wild-type sequence is shown above. The shaded areas correspond to deletions. Point mutations are shown in ▪.

Figure 5.

A malignant gastric GIST of spindle cell type shows plump spindle cells with conspicuous mitotic activity. Hematoxylin and eosin, original magnification ×482.

Figure 6.

The malignant GIST shown in Figure 3 ▶ is strongly immunoreactive for CD117 (left), but negative for α-smooth muscle actin; a vessel wall is positive (right). ABC immunoperoxidase, original magnification ×411.

Figure 7.

Analysis of the PCR amplification products of the exon 11 of c-kit in malignant GISTs. Note the different size mutant bands in different cases. Lane 1: Case 20. Lane 2: Case 34. Lane 3: Case 32. Lane 4: Case 21. Lane 5 : Case 27. Lane 6: Case 42. Lane 7: Case 30. Lane 8: Placenta (germline), Lane 9: Negative control. The samples are flanked by PhiX174 DNA/HinfI markers (Promega, Madison, WI). Arrows indicate 200- and 151-bp fragments. 5% polyacrylamide gel electrophoresis.