This study is the first to report c-kit
gene mutations in primary adenoid cystic carcinoma of the salivary gland. Activating mutations in the c-kit
gene have been shown in a spectrum of human tumors including kinase domain mutations in mast cell neoplasms and seminomas and extracellular and transmembrane mutations in acute myeloid leukemia and myeloproliferative disorders. In gastrointestinal stromal tumors, 65–92% of tumors are reported to harbor KIT
-activating mutations, the majority of which are localized to the juxtamembrane region involving exon 11.18,19
The majority of exon 11 mutations are clustered within the classic hotspot region of the 5′ end involving codons 550–560, however, a second hot spot at the 3′ end involving codons 576–590 has been described by Antonescu et al.17
These include frame deletions of one to several codons (typically involving codons 557–560), point mutations and internal tandem duplications (typically involving the 3′ end). In our study, point mutations in exon 11 were most frequently detected and were clustered in the 5′ and 3′ regions of exon 11. The types of point mutations were heterogeneous; however, substitutions similar to those described in gastrointestinal stromal tumors were detected including Leu576Phe in exon 11 and substitutions involving codons 551 and 558 (Pro551-Leu and Lys 558Glu).17,20,21
Novel exon 11 mutations detected in our study included Glu562Val, His580-Tyr, Phe584Leu and Phe591Leu.
Mutations in exons 9, 13 and 17 were less frequently detected than exon 11 mutations. These are considered rare in gastrointestinal stromal tumors with a reported frequency of less than 10%, but are seen more commonly in hematopoietic malignancies and germ cell neoplasms.22-24
Exon 13 mutations tend to occur in the vicinity of codon 642, with the 1945 A>G substitution leading to Lys642-Glu mutation being the most common.22
We detected the variant Val643Ala mutation observed in gastrointestinal stromal tumors. In addition, exon 17 mutations preferentially involve codon 822 with the 2487T>A substitution being most frequent.22
We detected a variant mutation in adenoid cystic carcinoma consisting of 2486T>A substitution leading to Asn822Ser. Additional novel mutations included Asp496Val and Asn512Asp in exon 9, Ser639Pro in exon 13, Leu813Pro in exon 17. We did not detect any insertions or duplications in exon 9, as have been reported involving codons 502–503 AY and 506–508 FAF.17,23-25
In two patients, we detected more than one mutation in exon 11. In addition, in two cases with exon 11 mutations, we detected additional mutations in exons 9 and 17, respectively. Interestingly, in one patient whose primary tumor contained multiple point mutations in exons 11 and 17 (Lys558Glu, Glu562Val, Phe591Leu and Leu813Pro), we detected a different mutation in exon 9 (Asp496Val) in the cervical level IIA lymph node metastasis. In general, involvement of different c-kit
domains is mutually exclusive; however, our results are in keeping with a few studies, which have reported multiple c-kit
mutations within the same tumor.26-28
Similar to our study, Andersson et al27
found multiple KIT
mutations involving different exons and also loss or addition of mutations in the tumor metastases compared with the primary tumors. Sakurai et al28
likewise identified different mutations in different samples from the same patient. These findings demonstrate the heterogeneity of KIT
mutations found within cells of the same tumor population.
Our results differ from four previous studies that did not detect KIT
mutations in adenoid cystic carcinomas of the salivary gland and lung.4,5,14-16
These studies used direct PCR sequencing techniques, which is by far, the most widely used; however, direct PCR sequencing lacks significant sensitivity compared to subcloning of the DNA products, because of the heterogeneous DNA from both normal and tumor samples. The high percentage of mutations detected in our study may be attributed to the increased sensitivity obtained through cloning of PCR products and selection of multiple colonies per each exon for DNA sequencing. The cloning selection allowed us to sequence the single species of DNA fragment for each sequencing reaction.
Our study demonstrates the presence of c-kit
gene mutations in adenoid cystic carcinomas. The functional significance of these mutations for KIT protein remains to be determined through many cell biology studies. Even less is known about how these mutations are related to potential treatment. There is conflicting data regarding the function of tyrosine kinase inhibitor treatment in adenoid cystic carcinoma. The results of Hotte et al29
showed no evidence of objective response among 15 patients treated with imatinib mesylate. Pfeffer et al30
and Ochel et al31
similarly showed no therapeutic response of locally advanced or metastatic adenoid cystic carcinomas to imatinib. These studies assessed response to tyrosine kinase inhibitor treatment using tumors with strong c-kit immunohistochemical expression; however, they did not assess genetic alterations of the c-kit
gene. Lin et al15
observed an unexpected rate of progression of metastatic adenoid cystic carcinoma during 2–3 weeks of treatment with imatinib in five tumors that contained no detectable c-kit
mutations. Based on their results, the authors concluded that use of imatinib to treat cancers without c-kit
mutations should be approached with caution. However, preliminary results of a phase II multicenter trial by Faivre et al32
have demonstrated that imatinib may have, at least in a subset of patients, antitumor activity in adenoid cystic carcinoma. Alcedo et al33
also reported two successfully treated cases of adenoid cystic carcinoma of the salivary gland with imatinib. Further studies are needed to investigate the type of KIT
mutations harbored by responders compared to nonresponders, as this may be of prognostic value in assessing response to tyrosine kinase inhibitor treatment.
In summary, this study is first to report the presence of c-kit gene mutations in primary adenoid cystic carcinoma. These potential gain-of-function mutations in exon 11, and less frequently in exons 9, 13 and 17 may be involved in KIT overexpression in adenoid cystic carcinomas. Future studies in a larger series may be of value to determine the prognostic implications and benefits of KIT-directed therapy in tumors harboring such mutations.