(American Journal of Pathology. 2002;161:739-741.)
© 2002 American Society for Investigative Pathology
Apparent KIT Ser715 Deletion in GIST mRNA Is Not Detectable in Genomic DNA and Represents a Previously Known Splice Variant of KIT Transcript
Jerzy Lasota,
Janusz Kopczynski,
Mourad Majidi and
Markku Miettinen
Armed Forces Institute of Pathology Washington, DC
Maarit Sarlomo-Rikala
Haartman Institute Helsinki, Finland
To the Editor-in-Chief:
Gain-of-function mutations leading into ligand-independent activation of KIT and having a transforming effect in vitro have been documented in gastrointestinal stromal tumors (GISTs).1
A majority of these mutations affects juxtamembrane KIT domain, although mutations in extracellular and kinase domains have also been reported in the subsets of GISTs.2–4
However, only one type of KIT mutation can be identified in any given tumor. The presence of two different somatic mutations in one tumor was reported once5
and seems to be an extremely rare event since has not been seen in other studies.2,4
Recently, an RNA-based study found deletions resulting in loss of Lys704 and Asn705 in the first KIT kinase domain (exon 14) and loss of Ser715 in the interkinase KIT domain (exon 15) in GISTs carrying mutations of juxtamembrane KIT domain.6
We have selected DNA and RNA samples from KIT-positive GISTs (74 samples from 69 patients) to evaluate molecular alterations in KIT exons 14 and 15 using PCR and RT-PCR amplification. There were 30 gastric, 16 small intestinal, 7 colonic, and 6 rectal primary tumors, 11 recurrent, and 4 tumors of unknown origin. KIT exon 11 and exon 9 mutations were present in 42 and 9 samples, respectively. Direct sequencing of exon 14 and 15 showed wild-type KIT sequences in 31 and 47 analyzed samples respectively. Similarly, one peak consistent with the presence of wild-type KIT only was seen in 13 cases analyzed by capillary gel electrophoresis (Figure 1A)
. However, capillary gel electrophoresis of the RT-PCR products revealed the presence of two peaks in 16 of 21 analyzed tumors (Figure 1B)
. Direct sequencing of the PCR products confirmed the presence of wild-type KIT and another, 3 bp shorter KIT transcript lacking Ser715 (Figure 1C)
. The ratio between the two forms of KIT transcripts was unequal with preferential expression of the wild-type KIT. This observation and a previously published studies on different types of KIT isoforms in normal and malignant hematopoietic cells7
clearly indicate that the deletion of Ser715 recently reported by Anderson and co-authors6
does not represent a somatic mutation involved in GISTs tumorigenesis, but rather a splice variant of KIT transcript.
View larger version (54K):
[in this window]
[in a new window]
|
Figure 1. Capillary gel electrophoresis fragment analysis and direct sequencing of KIT PCR amplification products. PCR amplification of exon 15 genomic sequences (A) and RT-PCR amplification of exon 14/15 junctional sequences (B). Blue peaks represent PCR products. Red peaks indicate molecular size markers, values in bp are shown below. Arrow indicates KIT Ser715-. Direct sequencing of RT-PCR product from B shows presence of KIT Ser715 +/- (C). The horizontal arrow indicates shift of allelic sequences.
|
|
Lack of mutation in exon 14 in our studies would not exclude the possibility that deletion of Lys704 and Asn705 could be a rare, possibly secondary, random molecular change. To investigate the relationship of alternative splicing between exon 14 and 15 and exon 11 pathological changes in GISTs, mutant, and wild-type KIT transcripts were amplified separately in three cases using the allele-specific primers from exon 11 and the common primer from exon 15. The wild-type KIT allele was amplified by a primer homologous to the deleted sequences, while the mutant KIT allele was amplified by a primer homologous to specific sequences created by the deletion. RT-PCR amplification of the KIT exon 11–15 generated products of 538–541 bp and 529–531 bp for wild-type and mutant KIT, respectively. Because separation of 3 bp different fragments in this size range is beyond resolution of capillary gel electrophoresis, a five cycle-long semi-nested PCR amplification was performed to obtain a shorter product. The specificity of this RT-PCR has been confirmed by amplification of KIT Gly-Asn-Asn-Lys510–513 isoforms using forward exon 9 primer and allele-specific reverse primers. In this case, smaller size RT-PCR products fractionated by capillary gel electrophoresis revealed predicted allele-specific sizes. Using this strategy, we have detected KIT Ser715- and KIT Gly-Asn-Asn-Lys510–513+ transcripts in GISTs from the wild-type and mutant KIT mRNA therefore supporting a naturally occurring variation in the splicing mechanism.
Two types of KIT isoforms have been reported, KIT Gly-Asn-Asn-Lys510–513+/- alternatively splices 12 bp located immediately downstream to the extracellular KIT domain following exon 9 sequences and KIT Ser715+/- alternatively splices first 3 bp of exon 15, an interkinase KIT domain.7,8
Expression of these isoforms has been documented in normal human hematopoietic cells, leukemic cell lines, acute myeloid leukemia blasts, and GISTs.6–8
However, no evidence has been found suggesting that up-regulation of KIT isoforms contributes to the neoplastic process.7,8
More recent studies showed that KIT Gly-Asn-Asn-Lys- and Gly-Asn-Asn-Lys+ differ in activation of downstream KIT signaling pathways and show different transforming activity when expressed in NIH3T3 cells; only cells expressing the Gly-Asn-Asn-Lys- isoform were tumorigenic in nude mice.9
Modification of KIT in GISTs by alternative splicing events involving exon 9 and exon 15 may contribute to different biological function of mutant KIT and should be further studied using in vitro and in vivo models.
Authors’ Note: The opinions and assertions contained herein are the expressed views of the authors and are not to be construed as official or reflecting the views of the Departments of the Army or Defense. This study was partly supported by the American Registry of Pathology.
References
- Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, Ishiguro S, Kawano K, Hanada M, Kurata A, Takeda M, Tunio GM, Matsuzawa Y, Kanakura Y, Shinomura Y, Kitamura Y: Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 1998, 279:577-580[Abstract/Free Full Text]
- Lux ML, Rubin BP, Biase TL, Chen C-J, Maclure T, Demetri G, Xiao S, Singer S, Fletcher CDM, Fletcher JA: KIT extracellular and kinase domain mutations in gastrointestinal stromal tumors. Am J Pathol 2000, 156:791-795[Abstract/Free Full Text]
- Lasota J, Wozniak A, Sarlomo-Rikala M, Rys J, Kordek R, Nassar A, Sobin LH, Miettinen M: Mutations in exons 9 and 13 of KIT gene are rare events in gastrointestinal stromal tumors. Am J Pathol 2000, 157:1091-1095[Abstract/Free Full Text]
- Rubin BP, Singer S, Tsao C, Duensing A, Lux ML, Ruiz R, Hibbard MK, Chen C-J, Xiao S, Tuveson DA, Demetri GD, Fletcher DMF, Fletcher JA: KIT activation is a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res 2001, 61:8118-8121[Abstract/Free Full Text]
- Sakurai S, Oguni S, Hironaka M, Fukayama M, Morinaga S, Saito K: Mutations in c-kit gene exons 9 and 13 in gastrointestinal stromal tumors among Japanese. Jpn J Cancer Res 2001, 92:494-498[Medline]
- Andersson J, Sjögren H, Meis-Kindblom JM, Stenman G, Åman P, Kindblom L-G: The complexity of KIT gene mutations and chromosome rearrangements and their clinical correlation in gastrointestinal stromal (pacemaker cell) tumors. Am J Pathol 2002, 160:15-22[Abstract/Free Full Text]
- Crosier PS, Ricciardi ST, Hall LR, Vitas MR, Clark SC, Crosier KE: Expression of isoforms of the human receptor tyrosine kinase c-kit in leukemic cell lines and acute myeloid leukemia. Blood 1993, 82:1151-1158[Abstract/Free Full Text]
- Piao X, Curtis JE, Minkin S, Minden MD, Bernstein A: Expression of the Kit and Kit A receptor isoforms in human acute myelogenous leukemia. Blood 1994, 83:476-481[Abstract/Free Full Text]
- Caruana G, Cambareri AC, Ashman LK: Isoforms of c-kit differ in activation of signaling pathways and transformation of NIH3T3 fibroblasts. Oncogene 1999, 18:5573-5581[Medline]
This article has been cited by other articles:
|
|
|
|
 
V. Munugalavadla, L. C. Dore, B. L. Tan, L. Hong, M. Vishnu, M. J. Weiss, and R. Kapur
Repression of c-Kit and Its Downstream Substrates by GATA-1 Inhibits Cell Proliferation during Erythroid Maturation
Mol. Cell. Biol.,
August 1, 2005;
25(15):
6747 - 6759.
[Abstract]
[Full Text]
[PDF]
|
|
|
