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• W2109508638 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. 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. Gastrointestinal stromal tumor (GIST) is the designation for a major subset of mesenchymal tumors of the gastrointestinal tract. These tumors differ from ordinary leiomyomas and leiomyosarcomas histologically, immunohistochemically and genetically. GISTs are typically more cellular than ordinary leiomyomas and are composed of spindle or epithelioid-appearing cells. Immunohistochemically they are typically positive for CD34, the hematopoietic progenitor cell antigen, but negative for desmin, in contrast to leiomyomas and leiomyosarcomas.1Mikhael AI Bacchi CE Zarbo RJ Ma CK Gown AM CD34 expression in stromal tumors of the gastrointestinal tract.Appl Immunohistochem. 1994; 2: 89-93Google Scholar, 2van de Rijn M Hendrickson MR Rouse RV The CD34 expression by gastrointestinal stromal tumors.Hum Pathol. 1994; 25: 766-771Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 3Monihan JM Carr NJ Sobin LH CD34 immunoexpression in stromal tumours of the gastrointestinal tract, and in mesenteric fibromatoses.Histopathology. 1994; 25: 469-473Crossref PubMed Scopus (113) Google Scholar, 4Miettinen M Virolainen M Sarlomo-Rikala M Gastrointestinal stromal tumors: value of CD34 antigen in their identification and separation from true leiomyomas and schwannomas.Am J Surg Pathol. 1995; 19: 207-216Crossref PubMed Scopus (505) Google Scholar, 5El-Rifai W Sarlomo-Rikala M Miettinen M Knuutila S Andersson LC DNA copy number losses in chromosome 14: an early change in gastrointestinal stromal tumors.Cancer Res. 1996; 56: 3230-3233PubMed Google Scholar, 6Sarlomo-Rikala M El-Rifai W Andersson L Miettinen M Knuutila S Different patterns of DNA copy number changes in gastrointestinal stromal tumors, leiomyomas and schwannomas.Hum Pathol. 1998; 29: 476-481Abstract Full Text PDF PubMed Scopus (89) Google Scholar Recently, the expression of the c-kit proto-oncogene has been shown in GISTs.7Hirota 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, 8Kindblom LG Remotti HE Aldenborg F Meis-Kindblom JM Gastrointestinal pacemaker cell tumor (GIPACT): gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal.Am J Pathol. 1998; 152: 1259-1269PubMed Google Scholar, 9Sarlomo-Rikala M Kovatich AJ Barusevicius A Miettinen M CD117: A sensitive marker for gastrointestinal stromal tumors that is more specific than CD34.Mod Pathol. 1998; 11: 728-734PubMed Google Scholar, 10Sircar K Hewlett BR Riddell RH Most gastrointestinal stromal tumors arise from interstitial cells of Cajal (Abstract).Mod Pathol. 1998; 11: 71AGoogle Scholar The c-kit gene encodes for a receptor for a growth factor termed stem cell factor. The c-kit gene product (stem cell factor receptor) contains an internal tyrosine kinase component and regulates cell growth and survival.11Vliagoftis H Worobec AS Metcalfe DD The proto-oncogene c-kit and c-kit ligand in human disease.J Allerg Clin Immunol. 1997; 100: 435-440Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 12Yarden Y Kuang WS Yang-Fend T Coussens L Munemitsas S Dull TJ Human proto-oncogene kit, a new cell surface-receptor tyrosine kinase for an unidentified ligand.EMBO J. 1987; 6: 3341-3351Crossref PubMed Scopus (1333) Google Scholar, 13Williams DE Eisenman J Baird A Rauch C van Ness K March CJ Park LS Martin U Mochizuki DY Boswell HS Burgess GS Cosman D Lyman SD Identification of a ligand for the c-kit proto-oncogene.Cell. 1990; 63: 167-174Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 14Zsebo KM Williams DA Geissler EN Broudy WC Martin FH Atkins HL Hsu R-Y Birkett NC Okino KH Murdock DC Jacobsen FW Langley KE Smith KA Takeishi T Cattanach BM Galli SJ Suggs SV Stem cell factor is encoded at the SI locus of the mouse and is ligand for the c-kit tyrosine kinase receptor.Cell. 1990; 63: 214-224Google Scholar Cell types in which c-kit expression plays a functionally important role include germ cells, melanocytes, hematopoietic stem cells, mast cells, and the interstitial cells of Cajal, the gastrointestinal pacemaker cells.15Tsuura Y Hiraki H Watanabe K Igarashi S Shimamura K Fukuda T Suzuki T Seito T Preferential localization of c-kit product in tissue mast cells, basal cells of skin, epithelial cells of breast, small cell lung carcinoma and seminoma/dysgerminoma in human: immunohistochemical study of formalin-fixed, paraffin-embedded tissues.Virchows Arch. 1994; 424: 135-141Crossref PubMed Scopus (206) Google Scholar, 16Maeda H Yamagata A Nishikawa S Yoshinaga K Kobayshy S Nishi K Nishikawa S Requirement of c-kit for development of intestinal pacemaker system.Development. 1992; 116: 369-375PubMed Google Scholar, 17Sanders KM A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract.Gastroenterology. 1996; 111: 492-515Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar A specific mutation in the tyrosine kinase domain of c-kit has been found in mast cell neoplasms.18Nagata H Worobec AS Oh CK Chowdhury BA Tannenbaum S Suzuki Y Metcalfe DD Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.Proc Natl Acad Sci USA. 1995; 92: 10560-10564Crossref PubMed Scopus (809) Google Scholar Mutations between the transmembrane and tyrosine kinase domains in exon 11 of c-kit have been shown in GISTs7Hirota 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, 19Nishida T Hirota S Taniguchi M Hashimoto K Isozaki K Nakamura H Kanakura Y Tanaka T Takabayshi A Matsuda H Kitamura Y Familial gastrointestinal stromal tumours with germline mutation of the KIT gene.Nat Genet. 1998; 19: 323-324Crossref PubMed Scopus (507) Google Scholar and in a mast cell leukemia cell line.20Furitsu T Tsujimura T Tono T Ikeda H Kitayama H Koshimizu U Sugahara H Butterfielf JH Ashman LK Kanayama Y Matsuzawa Y Kitamura Y Kanakura Y Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.J Clin Invest. 1993; 92: 1736-1744Crossref PubMed Scopus (749) Google Scholar The c-kit mutations in GISTs have been shown to lead to ligand-independent activation of the tyrosine kinase of c-kit and have a tumor promoting effect in vitro.7Hirota 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 The c-kit mutation status of exon 11 have been studied in relatively few GISTs (five sporadic and three familial tumors) and several questions remain open. Unknown are the possible relationship of c-kit expression and the mutations, the relationship of the mutations and malignancy, and the possible specificity of the mutations for GISTs, as previously suggested.7Hirota 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 In this study, we further examined the c-kit expression and mutation status of exon 11 of c-kit in a series of benign and malignant GISTs and smooth muscle tumors. The results showed a consistent c-kit expression in GISTs with or without detectable mutations. The mutations occur preferentially in malignant versus benign GISTs and do not occur in leiomyomas and leiomyosarcomas. These observation suggest that the mutations in the exon 11 of c-kit might represent useful molecular genetic markers for malignant GISTs. Nineteen benign and 24 malignant or potentially malignant gastrointestinal stromal tumors (GISTs) and three esophageal leiomyomas and 11 leiomyosarcomas from different sites were obtained from the files of the Haartman Institute of the University of Helsinki, Helsinki, Finland. The tumors were immunohistochemically analyzed for CD34, CD117 (the c-kit proto-oncogene protein product), α-smooth muscle actin, desmin, and S100-protein. Immunohistochemistry was performed by using the avidin-biotin peroxidase complex system and diaminobenizidine as the chromogen, as previously described.9Sarlomo-Rikala M Kovatich AJ Barusevicius A Miettinen M CD117: A sensitive marker for gastrointestinal stromal tumors that is more specific than CD34.Mod Pathol. 1998; 11: 728-734PubMed Google Scholar Negative and positive controls were included in each run. The primary antibodies, pretreatments, and dilutions are shown in Table 1.Table 1Monoclonal Antibodies and Antisera Used in this StudyMolecule/antibodyClonePretreatment, Antibody dilutionSourceCD117 (c-kit)polyclonalMW, 1:400Santa-Cruz Biotechnology, Santa Cruz, CACD34HPCA-1Pepsin, 1:50Becton-Dickinson, Mt View, CAα-smooth muscle actin1A4No treatment, 1:1600Sigma, St. Louis, MoDesminD33No treatment, 1:50Dako Corp., Carpinteria, CAS100-proteinpolyclonalNo treatment, 1:1600DakoMW, microwave-based antigen retrieval with citrate buffer; Pepsin, pepsin digestion, 30 minutes at 37°C. Open table in a new tab MW, microwave-based antigen retrieval with citrate buffer; Pepsin, pepsin digestion, 30 minutes at 37°C. DNA for the PCR amplification was obtained from fresh tissue by standard phenol-chloroform-isoamylalcohol extraction and from FFPE tissues using a previously published procedure.21Lasota J Franssila K Koo CH Miettinen M Molecular diagnosis of mantle cell lymphoma in paraffin-embedded tissue.Mod Pathol. 1996; 9: 361-366PubMed Google Scholar The quality of DNA templates was verified by PCR amplification of a 268-bp fragment of genomic DNA as previously described.22Greer CE Peterson SL Kiviat NB Manos MM PCR amplification from paraffin-embedded tissues: effects of fixative and fixation time.Am J Clin Pathol. 1991; 95: 117-124PubMed Google Scholar A PCR assay was developed to amplify exon 11 of c-kit. The forward primer CK10.4 (5′-CCA GAG TGC TCT AAT GAC TG-3′) and the reverse primer CK 11.2 (5′-ACT CAG CCT GTT TCT GGG AAA CTC-3′) were designed based on a previously published sequence of the human c-kit gene.23Andre C Hampe A Lachaume P Martin E Wang XP Manus V Hu WX Galibert F Sequence analysis of two genomic regions containing the KIT and FMS receptor tyrosine kinase genes.Genomics. 1997; 39: 216-226Crossref PubMed Scopus (51) Google Scholar This PCR assay (30 cycles) yielded amplification products of 192 bp. The PCR reaction conditions were the standard ones recommended by Perkin Elmer (Norwalk, CT). The annealing temperature was 56°C. The PCR products were size fractionated on 5% polyacrylamide gels and stained with ethidium bromide. The PCR products were purified from the gels, treated with Klenow DNA polymerase (Boehringer Mannheim, Indianapolis, IN), blunt end-ligated into pBSK+ phagemid (Stratagene, La Jolla, CA), and transfected into DH5α-competent cells (Gibco-BRL, Gaithersburg, MD). White colonies obtained from the plating on IPTG/X-gal selective medium (Gibco-BRL) were picked and screened by PCR as described above to confirm the presence of the appropriate inserts. Double-stranded DNA templates were prepared from randomly selected recombinants using Qiagen plasmid kit (Qiagen Inc., Chatsworth, CA) and sequenced on a 373 DNA-sequencer (Applied Biosystems, Foster City, CA). Five to nine clones were sequenced per case using the M13 forward primer. The subsets of clones that revealed consistent nucleotide differences from the germline were sequenced using the reverse primers. To prevent PCR contamination, standard precautions were undertaken. To exclude Taq-polymerase error as the source of clonal variation, placental DNA was amplified, cloned, and sequenced as described above. Computer analysis of the DNA sequences was performed using the Lasergene software (DNASTAR, Madison, WI) in connection with the data of the GenBank 107/EMBL 54 database (July 1998 edition). Demographic data and clinical and histological features of all GISTs analyzed in this study have been summarized in Table 2, Table 3. There were 25 gastric, 12 small intestinal, and 2 rectal primary tumors. Five intraabdominal recurrences and 2 liver metastases were also analyzed from a total of 43 patients. Two different lesions from each of three patients were analyzed.Table 2Clinical Data and c-kit Mutations of the Benign GISTs Analyzed in this StudyCaseAge/SexLocationCell typeTumor size or maximum diameter (cm)Status and follow-up in monthsc-kit mutant bandSequencing of exon 11 of c-kit168 /MStomachSpindle2.0NED 27−Not done266 /MStomachSpindle3–4−Not done369 /MStomachEpithelioid3–4−Not done463 /MStomachSpindle3.5−Not done564 /FStomachSpindle3.6NED 21−Wild-type660 /FStomachSpindle4× 4× 3−Wild-type757 /MStomachSpindle4NED 56−Not done870 /FStomachSpindle*Case 8 showed 2 mitoses/10 HPF and case 12 showed 3 mitoses/10 HPF. All other cases showed ≤1 mitosis/10 HPF.4.5× 4.5× 3.5NED 25−Wild-type940 /MStomachEpithelioid5NED 34−Wild-type1033 /MStomachSpindle5× 3.5× 3.5NED 44+Deletion1161 /FStomachEpithelioid5× 5NED 35−Wild-type1279 /FStomachSpindle*Case 8 showed 2 mitoses/10 HPF and case 12 showed 3 mitoses/10 HPF. All other cases showed ≤1 mitosis/10 HPF.8NED 58−One point mutation1345 /FStomachSpindle9NED 24−Two point mutations1469 /MSmall bowelSpindle2.5× 2.0× 2.0NED 8−Not done1535 /FSmall bowelSpindle3–4−Wild-type1657 /MSmall bowelEpithelioid4NED 54−Wild-type1780 /MSmall bowelSpindle4.5NED 31−Not done1860 /MSmall bowelSpindle6× 6× 6−Not done1952 /MSmall bowelSpindle7 × 7× 6.5NED 27−Not doneNED, no evidence of disease.* Case 8 showed 2 mitoses/10 HPF and case 12 showed 3 mitoses/10 HPF. All other cases showed ≤1 mitosis/10 HPF. Open table in a new tab Table 3Clinical Data and c-kit Mutations of the Malignant and Potentially Malignant GISTs Analyzed in this StudyCaseAge/SexLocationCell typeTumor size or maximum diameter (cm)Mitoses/ 10 HPFClinical behavior, follow-up (months)c-kit mutant bandSequencing of exon 11 of c-kit20*Cases classified as potentially malignant.62 /MStomachSpindle152Died of lung cancer, 36 months. No evidence for IAR+Deletion21*Cases classified as potentially malignant.80 /FStomachEpithelioid5.5× 3.5× 1.7<1+Deletion22*Cases classified as potentially malignant.72 /MStomachEpithelioid11× 10× 3.5<1−Wild type2326 /FStomachEpithelioid6× 4.5× 2.518−Wild type2447 /FStomachSpindle10× 6× 511Omental seeding at present-ation. IAR at 15 months+Deletion2557 /MStomachSpindle9× 7.5× 712Mediastinal metastasis at presentation−Not done2670 /MStomachSpindle11× 518Omental seeding at presentation. IAR at 25 months+Deletion+ PM2769 /FStomachSpindle129NED 6 months+Deletion2844 /MStomachEpithelioid17 × 10× 59IAR at 25 months−Wild type2944 /MStomachSpindle176−Deletion3063 /MStomachSpindle19× 12× 78+D+ 3 PM3141 /MStomach39× 1830Alive with disease, 16 months−Not done3250 /MSBSpindle6.5× 4.5× 3.411Liver metastasis at presentation+Deletion3368 /MSBSpindle7× 68IAR at 16 months−Not done3460 /MSBSpindle76IAR at 12 months DOD 24 months+Deletion3564 /FSBSpindle12–15>20DOD 15 months−Wild type3654 /MSB+ mesenterySpindle15× 13× 615IAR 8 months−Wild type3785 /MSBSpindle30× 30× 515DOD 16 months−PM3863 /MRectumSpindle713NED 20 months−PM3973 /MRectumSpindle10× 9× 810+Deletion4062 /FIAR (2 yrs)Spindle2 kg, multiple tumorsIAR 2 and 3 years after a gastric GIST+Deletion4156 /FIAR (5 yrs)Spindle>20 cmIAR 4 and 5 years after a total gastrectomy for gastric malignant GIST−Wild type4259 /FLiver metastasisSpindle8 cmLiver metastasis 4 years after malignant gastric GIST+Deletion4364 /MLiver metastasisSpindle6 cmLiver metastasis 6 months after SB GIST−Not doneDOD, died of disease; NED, no evidence of disease; SB, small bowel; IAR, intra-abdominal recurrence; D, deletion; PM, point mutation.* Cases classified as potentially malignant. Open table in a new tab NED, no evidence of disease. DOD, died of disease; NED, no evidence of disease; SB, small bowel; IAR, intra-abdominal recurrence; D, deletion; PM, point mutation. Nineteen GISTs were classified as histologically benign based on low, if any, mitotic activity. All but two cases showed < 1 mitosis per 10 high power fields (HPF), one case showed 2 mitoses/10 HPF, and another 3 mitoses/10 HPF. The tumor size did not exceed 10 cm in the group of spindle cell tumors or 5 cm in the group of epithelioid tumors. Follow-up available in 13 cases did not show intra-abdominal recurrences or metastases in any of the cases. Histologically, 15 cases showed exclusively or predominantly a spindle cell pattern typically showing high cellularity, frequent perinuclear vacuoles, a rich capillary vascular pattern, and a general lack of cytoplasmic eosinophilia (Figure 1). Epithelioid cytologic features with large cytoplasmic cells and distinct cell borders were seen exclusively or predominantly in four cases. All cases, irrespective of morphological subtype and location, were immunohistochemically positive for CD117 (Figure 2). Most cases (74%) were positive for CD34, whereas a minority of the tumors (32%) expressed α-smooth muscle actin. All cases were negative for desmin except two epithelioid GISTs that showed focal desmin-reactivity seen in less than 10% of the tumor cells. All cases were negative for S100-protein.Figure 2A 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of the PCR amplification products of the critical region in exon 11 of c-kit revealed a mutant band in one of 19 benign GISTs (Case 10). Sequence analysis showed an in-frame deletion of 6 bp involving codons 556–558. The remaining 18 benign GISTs revealed only one wild-type band each. Wil-type bands from 11 cases were cloned and sequenced. In two benign GISTs (Case 12 and 13) sequencing showed mutations at codon 560 (Figure 3). Predicted amino acid sequences of the mutant c-kit are shown in Figure 4.Figure 4Predicted 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 ▪.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Twenty-one GISTs were classified as histologically malignant based on high mitotic activity (> 4 mitoses/10 HPF). The three tumors classified as potentially malignant showed a low mitotic activity but were large tumors and included a 15-cm spindle cell tumor from the stomach and two large epithelioid gastric tumors of 5.5 cm and 11 cm diameter. Follow-up available in 15 cases of malignant or potentially malignant GISTs showed intraabdominal recurrences, metastases, or death from tumor in 13 of them. Histologically, 19 tumors had a predominant spindle cell pattern (Figure 5), and four were epithelioid. CD117-immunoreactivity was shown in all cases (Figure 6), except in one (Case 26). The majority of cases (83%) were positive for CD34 and α-smooth muscle actin was present in 6 cases (26%), usually focally. All cases were negative for desmin and S100-protein.Figure 6The 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of the PCR amplification products of exon 11 of c-kit showed smaller mutant bands, in addition to the wild-type bands, in 12 of the 24 (50%) malignant GISTs (Figure 7). Sequencing of the mutant bands revealed in-frame deletions of 6 to 21 bp (Figure 3). Wild-type bands from these cases showed germline sequences of the c-kit. The remaining 12 malignant GISTs revealed only wild-type bands, and eight of them were sequenced. Three of the eight cases showed c-kit mutations. Two cases (Cases 37 and 38) showed point mutations, one at the codon 557 and another at the codon 576. The third case (Case 29) revealed a 3-bp in-frame deletion involving codons 560 and 561. Predicted amino acid sequences of the mutant c-kit are shown in Figure 4. Among the malignant GISTs, c-kit mutations were seen in tumors of different sites including gastric, small intestinal, and rectal primary tumors. In three cases, two consecutive lesions of the same patient were analyzed. They included primary tumor and an intraabdominal recurrence in Cases 24 and 34 and two intra-abdominal recurrences in Case 40. In all cases, the banding pattern and sequences of the mutant bands remained identical in primary and recurrent lesions. The two esophageal leiomyomas and one small intestinal leiomyoma were histologically spindle cell neoplasms with distinctly eosinophilic cytoplasm, moderate cellularity, and lack of mitotic activity. All cases were positive for α-smooth muscle actin and desmin and negative for CD34 and CD117. The eleven leiomyosarcomas included six from the retroperitoneum, two from the thigh, one from the inferior vena cava and two metastatic uterine leiomyosarcomas, one from the buttock and another from the small intestine. Histologically these tumors showed intersecting fascicles of spindle cells with blunt-ended nuclei and variably eo- sinophilic cytoplasm. Significant mitotic activity was present in each case. All cases were positive for α-smooth muscle actin and 9 of 11 at least focally for desmin; all were negative CD117 and S100-protein. Two cases were focally positive for CD34; all other cases were negative. Analysis of the PCR-amplification products of exon 11 of c-kit of three leiomyomas and 11 leiomyosarcomas showed wild-type bands similar to those seen in placental DNA. Sequencing revealed 100% homology with the c-kit germline. No nucleotide differences were identified in ten randomly selected PCR products from the amplification of exon 11 of c-kit from placental DNA. This gives a Taq-polymerase error rate of less than 1 nucleotide difference in 1920 bp and practically ruled out Taq-polymerase error as the source of the nucleotide variation seen in the exon 11 of c-kit in GISTs. In this study, we examined the sequences and expression of c-kit in the spectrum of gastrointestinal stromal tumors (GISTs), including benign and malignant variants from different sites. The sequences of the c-kit were also evaluated in typical smooth muscle tumors. Previous molecular studies7Hirota 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 have revealed mutations between the transmembrane and tyrosine kinase domains in five GISTs. Specifically, the mutations were located within codons 550 to 560 in exon 11, and no mutations were detectable in other domains of c-kit cDNA. Therefore, the exon 11 sequences coding amino acids 550–582 were analyzed in the current study. Our series of 43 benign and malignant GISTs showed that mutations in exon 11 of c-kit occur predominantly in the malignant GISTs that were tumors with high mitotic activity. Furthermore, clinically malignant tumor behavior was evident in most cases by intra-abdominal recurrences, distant metastases, or both. Mutations in the exon 11 of c-kit were observed in 62% of these tumors. In contrast, only 3 benign GISTs (16%) showed mutations, suggesting that the mutations of exon 11 of c-kit may represent a genotypic marker with a correlation to malignancy. The c-kit mutations were seen in malignant GISTs of different sites including gastric, small intestinal, and rectal tumors, indicating that in respect to the c-kit mutation status, malignant GISTs appear molecularly similar. Lack of c-kit mutations in 12/24 malignant and potentially malignant GISTs suggests that the mutations in the exon 11 of c-kit are not the only mechanisms related to tumorigenesis and malignancy and that other molecular mechanisms explaining the differences between benign and malignant GISTs must exist. Such mechanisms may include mutations in other regions of the c-kit. Involve- ment of other genes is suggested by the greater number of the DNA copy number changes in malignant versus benign GISTs as observed in comparative genomic hybridization studies.5El-Rifai W Sarlomo-Rikala M Miettinen M Knuutila S Andersson LC DNA copy number losses in chromosome 14: an early change in gastrointestinal stromal tumors.Cancer Res. 1996; 56: 3230-3233PubMed Google Scholar Most c-kit mutations were found in the previously described hot spot region of exon 117Hirota 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 involving codons 550–560. Clustering of the c-kit mutations in the same region supports their specific biological significance. However, in two cases, an additional region including codons 567–576 was found to be affected by both a point mutation and a deletion in two different cases. These findings suggest the possibility that mutations can involve other areas of the exon 11 of c-kit than previously described. Analysis of the c-kit mutation pattern in consecutive lesions in 3 cases showed the persistence of the mutations that remained identical in the different recurrences. Therefore, the tumor-specific c-kit mutation pattern could be used as a potential marker of early recurrence or to trace minimal residual disease. Further studies are necessary to explore the tumor cell detection in such contexts. The c-kit mutations were never observed in benign leiomyomas or well-documented leiomyosarcomas (actin-positive, usually desmin-positive, CD34-negative, CD117-negative). Therefore, the c-kit mutations may also represent a genotypic lineage marker for GISTs. In this respect, our findings confirm and expand on the earlier reports on the absence of c-kit mutations in smooth muscle tumors.7Hirota 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 Other genetic changes found in GISTs but not in smooth muscle tumors include losses in the long arm of chromosome 14, as reported based on comparative genomic hybridization studies.5El-Rifai W Sarlomo-Rikala M Miettinen M Knuutila S Andersson LC DNA copy number losses in chromosome 14: an early change in gastrointestinal stromal tumors.Cancer Res. 1996; 56: 3230-3233PubMed Google Scholar, 6Sarlomo-Rikala M El-Rifai W Andersson L Miettinen M Knuutila S Different patterns of DNA copy number changes in gastrointestinal stromal tumors, leiomyomas and schwannomas.Hum Pathol. 1998; 29: 476-481Abstract Full Text PDF PubMed Scopus (89) Google Scholar The presence of the mutations in exon 11 of c-kit did not correlate with c-kit expression as determined by immunohistochemistry. Whereas virtually all GISTs, including benign and malignant tumors, expressed c-kit, mutations occurred mostly in malignant cases. Therefore, explanations other than mutations in exon 11 have to be considered for the source of high c-kit expression. The interstitial cells of Cajal in the gastrointestinal nervous system show constitutional expression of c-kit and have recently been suggested as ancestor cells for GISTs.7Hirota 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, 8Kindblom LG Remotti HE Aldenborg F Meis-Kindblom JM Gastrointestinal pacemaker cell tumor (GIPACT): gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal.Am J Pathol. 1998; 152: 1259-1269PubMed Google Scholar, 10Sircar K Hewlett BR Riddell RH Most gastrointestinal stromal tumors arise from interstitial cells of Cajal (Abstract).Mod Pathol. 1998; 11: 71AGoogle Scholar, 16Maeda H Yamagata A Nishikawa S Yoshinaga K Kobayshy S Nishi K Nishikawa S Requirement of c-kit for development of intestinal pacemaker system.Development. 1992; 116: 369-375PubMed Google Scholar, 17Sanders KM A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract.Gastroenterology. 1996; 111: 492-515Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar Therefore, c-kit expression in GISTs could merely reflect the constitutional c-kit expression that is maintained in the neoplastic state of tumor cells, which may be related to the Cajal cells or show differentiation toward them.8Kindblom LG Remotti HE Aldenborg F Meis-Kindblom JM Gastrointestinal pacemaker cell tumor (GIPACT): gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal.Am J Pathol. 1998; 152: 1259-1269PubMed Google Scholar, 10Sircar K Hewlett BR Riddell RH Most gastrointestinal stromal tumors arise from interstitial cells of Cajal (Abstract).Mod Pathol. 1998; 11: 71AGoogle Scholar In summary, we have evaluated a series of GISTs and typical smooth muscle tumors for the expression of c-kit protein and for the mutations in exon 11 of c-kit. The c-kit expression is consistent in both benign and malignant GISTs with and without the c-kit mutations. The c-kit mutations occur preferentially in malignant GISTs. The mutations seem to remain stable in recurrences, and could be useful tumor-specific markers. Leiomyomas and leiomyosarcomas show neither c-kit expression nor mutations, adding a new phenotypic and genotypic marker to distinguish these tumors from GISTs." @default.
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