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Apparent KIT Ser715 Deletion in GIST mRNA Is Not Detectable in Genomic DNA and Represents a Previously Known Splice Variant of KIT Transcript

2002; Elsevier BV; Volume: 161; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)64230-7

1525-2191

Jerzy Lasota, Janusz Kopczyński, Mourad Majidi, Markku Miettinen, Maarit Sarlomo‐Rikala,

Vascular Malformations and Hemangiomas

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).1Hirota 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: 577-580Crossref PubMed Scopus (3885) Google Scholar 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.2Lux 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: 791-795Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 3Lasota 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: 1091-1095Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 4Rubin 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: 8118-8121PubMed Google Scholar 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 once5Sakurai 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: 494-498Crossref PubMed Scopus (83) Google Scholar and seems to be an extremely rare event since has not been seen in other studies.2Lux 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: 791-795Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 4Rubin 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: 8118-8121PubMed Google Scholar 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.6Andersson 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: 15-22Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar 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 cells7Crosier 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: 1151-1158Crossref PubMed Google Scholar clearly indicate that the deletion of Ser715 recently reported by Anderson and co-authors6Andersson 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: 15-22Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar does not represent a somatic mutation involved in GISTs tumorigenesis, but rather a splice variant of KIT transcript. 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.7Crosier 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: 1151-1158Crossref PubMed Google Scholar, 8Piao 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: 476-481Crossref PubMed Google Scholar Expression of these isoforms has been documented in normal human hematopoietic cells, leukemic cell lines, acute myeloid leukemia blasts, and GISTs.6Andersson 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: 15-22Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 7Crosier 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: 1151-1158Crossref PubMed Google Scholar, 8Piao 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: 476-481Crossref PubMed Google Scholar However, no evidence has been found suggesting that up-regulation of KIT isoforms contributes to the neoplastic process.7Crosier 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: 1151-1158Crossref PubMed Google Scholar, 8Piao 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: 476-481Crossref PubMed Google Scholar 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.9Caruana G Cambareri AC Ashman LK Isoforms of c-kit differ in activation of signaling pathways and transformation of NIH3T3 fibroblasts.Oncogene. 1999; 18: 5573-5581Crossref PubMed Scopus (83) Google Scholar 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.