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The great majority of gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of the c-kit gene, which encodes KIT receptor tyrosine kinase. Most of the mutations are located at exon 11, but some are at exon 9 or at other exons. Mutation types at exon 11 vary, while most mutations at exon 9 are a particular duplication of Ala502Tyr503 (KIT-Dup-Ala502Tyr503). Recently a duplication of Ser501Ala502 (KIT-Dup-Ser501Ala502) at exon 9 has been reported in two cases of pediatric mastocytosis and one case of adult mast cell leukemia. Although KIT-Dup-Ser501Ala502 had not been reported in GISTs, we found two GIST cases possessing the mutation in 45 GIST cases with exon 9 c-kit gene mutations, among a total of approximately 500 GIST cases examined. In this report, we briefly summarize clinicopathological findings of the two cases, and characterize the biology of the mutation. When autophosphorylation of KIT-Dup-Ser501Ala502 was examined by transient transfection of c-kit cDNA with Dup-Ser501Ala502 into CHO-K1 cells, KIT-Dup-Ser501Ala502 was ligand-independently activating. The inhibitory effect of selective tyrosine kinase inhibitors, imatinib and nilotinib, on KIT-Dup-Ser501Ala502 was examined and compared with that of KIT-Dup-Ala502Tyr503. Imatinib efficiently inhibited constitutive activation of KIT-Dup-Ser501Ala502 at a concentration of 0.1 μM, whereas it inhibited that of KIT-Dup-Ala502Tyr503 at a concentration of 10 μM. Constitutive activation of KIT-Dup-Ser502Ala503 was not inhibited by nilotinib even at a concentration of 10 μM but that of KIT-Dup-Ala501Tyr502 was almost completely inhibited at a concentration of 1 μM. The results suggest that imatinib and nilotinib could be more effective on GISTs with KIT-Dup-Ser501Ala502 than those with KIT-Dup-Ala502Tyr503. In fact, a patient with KIT-Dup-Ser501Ala502 showed long-term stable disease with administration of the usual dose of 400 mg imatinib. Although mutation sites of KIT-Dup-Ser501Ala502 and KIT-Dup-Ala502Tyr503 are closely located, imatinib- and nilotinib-sensitive KIT-Dup-Ser501Ala502 are distinguishable from KIT-Dup-Ala502Tyr503.

We found a novel type germline mutation at exon 11 of the c-kit gene, which results in a substitution of Tyr to Cys at codon 553 of the c-kit gene product (KIT-Tyr553Cys), in a 68-year-old female patient with multiple gastrointestinal stromal tumors (GISTs). In the present study, we carried out mutational analysis in her family members to determine the carriers and characterized the mutation by introducing the corresponding mutation (murine KIT-Tyr552Cys) into expression vector possessing murine c-kit cDNA. Mutational analysis of peripheral blood leukocytes of her family members revealed that a 44-year-old son had the same mutation, but at present he had neither apparent symptoms nor images of multiple GISTs. By transfection with the expression vector possessing the murine mutant c-kit cDNA, interleukin-3-dependent Ba/F3 murine lymphoid cells started growing autonomously without any growth factors, indicating that the mutation was considered to be of gain-of-function. Imatinib, a small molecule of tyrosine kinase inhibitor, effectively inhibited autophosphorylation of KIT-Tyr552Cys. Nilotinib, another small molecule of the KIT inhibitor, also effectively inhibited autophosphorylation of KIT-Tyr552Cys. In fact, proliferation of Ba/F3 cells expressing KIT-Tyr552Cys was effectively inhibited by both imatinib and nilotinib. These findings indicate that the novel type human KIT-Tyr553Cys mutation is the cause of the present familial and multiple GISTs, and that both imatinib and nilotinib might effectively inhibit the growth of GISTs developing in the patients of this family.