Differential Expression of Metallothionein in Gastrointestinal Stromal Tumors and Gastric Carcinomas.код для вставкиСкачать
THE ANATOMICAL RECORD 294:267–272 (2011) Differential Expression of Metallothionein in Gastrointestinal Stromal Tumors and Gastric Carcinomas ELIZA TING-LI SOO,1 CHENG-TENG NG,1 GEORGE WAI-CHEONG YIP,1 CHUAY YENG KOO,1 MIN-EN NGA,2 PUAY-HOON TAN,3 AND BOON-HUAT BAY1* 1 Department of Anatomy, National University of Singapore, Singapore 2 Department of Pathology, National University of Singapore, Singapore 3 Department of Pathology, Singapore General Hospital, Singapore ABSTRACT Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that account for about 2% of gastric tumors. Metallothioneins (MTs) are multifunctional proteins associated with carcinogenesis and known to be coded by 10 functional MT genes. This study evaluated MT mRNA and protein expression in GISTs and compared the expression levels with gastric carcinomas. An immunohistochemical study of MT protein expression was performed in 15 GISTs (speciﬁcally located in the stomach) and 38 early stage gastric carcinomas. The percentage of cells stained and intensity of staining were determined. MT-2A mRNA expression was investigated in 6 GISTs and 6 early stage gastric carcinoma patients. All GISTs displayed positive nuclear immunostaining, with most GISTs having predominantly mildly stained nuclei (93.3%). On the other hand, 37 out of 38 gastric carcinoma cases were positively stained for nuclear MT with 24 cases (63.2%) exhibiting predominantly mild nuclear staining, 7 cases (18.4%) moderate nuclear staining, and 6 cases (15.8%) strong nuclear staining. Nuclear MT expression was found to be signiﬁcantly lower in GIST samples when compared with gastric carcinoma tissues based on the percentage stained and immunoreactive score. We then established that the MT-2A gene transcript was the most abundant MT isoform in MKN28 gastric cancer cells and analyzed its expression in GIST and gastric carcinoma tissues. We found that GISTs had signiﬁcantly lower MT-2A mRNA levels than gastric carcinoma tissues. Lower MT-2A gene expression and nuclear MT protein expression in GISTs when compared with gastric carcinomas may reﬂect their different underlying biology and divergent histogenesis. Anat Rec, 294:267–272, C 2010 Wiley-Liss, Inc. 2011. V Key words: metallothionein; MT-2A; GIST; gastric carcinoma Gastrointestinal stromal tumors (GISTs) are speciﬁc mesenchymal tumors that account for about 2% of gastric tumors and reported to be most common in the stomach (60–70%) followed by the small intestine (25– 35%) (Miettinen et al., 1998). Other examples of submucosal mesenchymal tumors of the gastrointestinal tract include leiomyosarcomas and gastrointestinal Kaposi’s sarcoma (Ponsaing et al., 2007). GISTs occur mainly in middle-aged and older people and are rarely found in patients below the age of 40 (Miettinen et al., 1999a). It C 2010 WILEY-LISS, INC. V immunohistochemistry; Grant sponsor: National Research Foundation, Singapore; Grant number: NMRC/TCR/001/NUS/2007. *Correspondence to: Boon-Huat Bay, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Blk MD10, S117 597, Singapore. Fax: þ65-6778 7643. E-mail: [email protected] Received 16 July 2010; Accepted 7 November 2010 DOI 10.1002/ar.21321 Published online 23 December 2010 in Wiley Online Library (wileyonlinelibrary.com). 268 SOO ET AL. has been reported that as a signiﬁcant number of GISTs are less than 2 cm and asymptomatic, these tumors are detected only incidentally during surgery for unrelated diseases (Miettinen et al., 1999a). GISTs comprise several distinct morphologic patterns including epithelioid, spindle cell, mixed and occasionally pleomorphic types (Miettinen and Lasota, 2006). The most speciﬁc diagnostic criterion to date for GIST is detection of c-KIT expression immunohistochemically (Hirota et al., 1998; Sarlomo-Rikala et al., 1998; Liegl-Atzwanger et al., 2010). C-KIT protein (CD117) expression has been reported to be 95% positive in GIST and suggested by the World Health Organization to be a deﬁning feature of GISTs (Ponsaing et al., 2007). Gene mutation of KIT or platelet-derived growth factor receptor alpha are reported to induce oncogenic signalling in the absence of their ligands, leading to activation of the PI3K-AKT and MEKMAPK pathways that promote tumorigenesis (Heinrich et al., 2003; Rossi et al., 2006; Liegl-Atzwanger et al., 2010). Most GISTs are positive for CD34 and heavy molecular weight caldesmon (Miettinen et al., 1999b; Miettinen et al., 2006). GISTs are also typically negative for S100-protein and rarely express desmin—an intermediate ﬁlament protein (Miettinen et al., 2000). Gastric carcinoma, an epithelial cancer, is the fourth most common cancer worldwide. It is the second cause of cancer mortality leading to 700,000 deaths annually (Parkin et al., 2005). The incidence of gastric carcinoma is reported to be highest in Asian countries, though timetrend studies have shown a decrease incidence of this cancer in many countries in Asia (Fock and Ang, 2010). Gastric carcinoma is a genetically heterogeneous disease associated with multiple carcinogenic pathways (Panani, 2008). In fact, the occurrence of gastric carcinoma in Asian population tends to mirror the seroprevalence rate of Helicobacter pylori infection although this is not always seen (Fock and Ang, 2010). Even though early gastric carcinomas have a favourable clinical outcome, advanced (unresectable or recurrent) cases have a poor prognosis, resulting in the call for new approaches to therapy such as a biomarker oriented strategy (Boku, 2010). One biomarker investigated is metallothionein (MT), which is known to be expressed in a cell-speciﬁc and tissue-speciﬁc manner in different types of tumors (Cherian et al., 2003). MTs are low molecular metal-binding proteins ﬁrst discovered in 1957 by Margoshes and Vallee (Margoshes and Vallee, 1957; Klaassen et al., 1999). There are at least 10 functional MT genes (MT-1A, 1B. 1E, 1F, 1G, 1H, 1X, 2A, 3, and 4 isoforms) encoding four MT proteins. As MTs are multifunctional proteins, they have been implicated in carcinogenesis and tumor progression in a variety of cancers (Coyle et al., 2002; Cherian et al., 2003; Thirumoorthy et al., 2007). In this study, we evaluated the expression of MT in GISTs and gastric carcinoma by immunohistochemical staining and realtime quantitative real-time polymerase chain reaction (RT-PCR) to investigate if there is differential expression of MT in tumors arising from histological layers of the stomach with different embryonic origins. MATERIALS AND METHODS Patients and Tumor Samples A total of 15 GISTs with speciﬁc location in the stomach and 38 early stage (Stage 1 and 2) gastric carcinoma cases were included in the study. These tissue samples were obtained from patients who had undergone surgery at the National University Hospital (NUH), and the use of tissues for research was approved by the Institutional Review Board. The histological diagnosis was made on haematoxylin and eosin (H&E) stained slides according to standard criteria. The tissues were ﬁxed in 10% formalin and embedded in parafﬁn for immunohistochemical studies. Immunostaining results of a panel of markers, viz., CD 117, CD34, S-100, desmin, and proliferating cell nuclear antigen (PCNA), were retrieved from the pathological data sheets of the GISTs. For gastric cancer, there were 26 intestinal type, 9 diffuse type, and 3 mixed type as categorized by the Lauren classiﬁcation. All the samples were harvested by pathologists from the Department of Pathology, NUH. Immunohistochemistry Immunohistochemistry on parafﬁn-embedded GISTs and gastric carcinomas performed using the Leica BondTM-Max System (Leica Microsystems, Wetzlar, Germany) with fully automated immunohistochemical staining. The primary MT antibody (Dako Corporation, Carpinteria, CA) was diluted by 100 times. The time for each run was 2 hr and 30 min. A drop of Permount mounting medium (Fisher-Scientiﬁc, Fair Lawn, NJ) was applied to each slide and mounted with a coverslip. Stained slides were scanned using ScanScope digital scanners (Aperio, Vista, CA). The scanned slides were then viewed and scored using the Aperio ImageScope to determine the intensity of immunostaining. MT immunopositivity was deﬁned as the presence of cytoplasmic and/or nuclear staining. The intensity of staining was scored as 0 (no detectable immunoreactivity), 1þ (mild staining), 2þ (moderate staining), and 3þ (strong staining). The following methods were used to quantitate nuclear staining: percentage stained and immunoreactive score (IRS). IRS was derived from the sum of all the products of each stained intensity with the corresponding percentage stained. Cell Culture Moderately differentiated human MKN28 gastric cancer cells (a gift from Professor Y. Ito, National Cancer Institute Singapore) were cultured in Roswell Park Memorial Institute medium (RPMI 1640) (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT) and 100 units/mL penicillin and 100 lg/mL streptomycin (Invitrogen, Carlsbad, CA) and maintained at 37 C in a 5% CO2 incubator. Immunocytochemistry A total of 5 104 MKN28 cells were seeded separately into Lab-TekTM 4-well chamber coverglass (Nalge Nunc International, Rochester, NY). Cells were ﬁxed with 4% paraformaldehyde. Primary mouse anti-horse antibody E9 ((Dako) at a dilution factor of 1:200 was added and incubated at 4 C, overnight. A negative control (without the addition of MT antibody) was included. Biotinylated, afﬁnity-puriﬁed secondary anti-mouse antibody at the same dilution factor was added for 1 hr at room temperature. The cells were washed, incubated for 1 hr at room 269 MT EXPRESSION IN GISTS AND GASTRIC CANCER temperature with ABC solution (Avidin DH and Biotinylated Horseradish Peroxidase H diluted in PBS-TX) (Vector Laboratories, Burlingame, CA) followed by the addition of 3,3-diaminobenzidene (DAB) mixture (including TBS and hydrogen peroxide) for 10 min. The cells were counterstained with Shandon’s Haematoxylin, rinsed in distilled water, dehydrated using alcohol and histoclear to remove alcohol. The glass cover slip was mounted onto a glass slide with Permount mounting medium and air dried. Quantitative RT-PCR RNA was reversed transcribed to cDNA using the Superscript III system (Invitrogen). For RT-PCR analysis, primers that target the eight functional MT-1 and MT-2 isoforms were adopted from Mididoddi et al. (1996). The house-keeping gene glyceraldehyde-3-phosphate-dehydrogenase (G3PDH) was included as a normalization control. The primers used for G3PDH were: forward 50 -GAA GGT GAA GGT CGG AGT CAA CG-30 ; reverse 50 -TGC CAT GGG TGG AAT CAT ATT GG -30 . The following RT-PCR conditions were used: 95 C for 15 min for denaturation, followed by 50 cycles of 94 C for 15 sec, 55 C for 40 sec and 72 C for 20 sec for annealing. The ﬂuorescence from SYBR Green was determined at the melting temperature of 50–60 C, 20 sec followed by cooling at 72 C, 30 sec. Quantiﬁcation was calculated using DCT, which is the difference between the target gene and G3PDH. The speciﬁcity of PCR products was further veriﬁed by agarose gel electrophoresis of the resulting PCR products. TissueScan Array GISTs and gastric carcinoma (1 Stage IA, 2 Stage IB, and 3 Stage II) tissues were selected from the TissueScan Gastroesophageal Tissue qPCR Panel I (OriGene, Rockville, MD). Each plate was removed from a 20 C refrigerator and allowed to warm to room temperature. A pre-mix was prepared and aliquoted into each of the 48 wells according to the manufacturer’s protocol. The plate was then covered with a new adhesive cover sheet and sealed tightly by pressing the cover around each well before placing on ice for 15 min to allow the cDNA to dissolve. SYBR Green dye was used as the reporter dye in this experiment. The speciﬁc MT-2A primers used were forward 50 -GGA TCC GAT CCC AAC TGC TCC TGC GCC-30 ; reverse 50 -CTC GAG TCA GGC GCA GCA GCT GCA CTT-30 . RT ﬂuorescent detection of PCR products was performed using Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA) with the following thermocycling conditions: 95 C for 15 min for one cycle, followed by 40 cycles of 94 C for 15 sec, 60 C for 30 sec, and 72 C for 60 sec. The CT values were obtained for further analysis. TABLE 1. Immunohistochemical staining of MT and a panel of markers speciﬁc for GIST S/No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Nuclear MT (%) Cyto MT (%) CD117 CD34 S100 Desmin 22.5 10 12.5 12.5 50 5 5 35 22.5 5 20 25 5 5 5 17.5 5 0 10 75 5 15 3.5 7.5 0 30 10 0 2.5 0 N P N N P P P N P P P P P P P P P N P P P P N P P P P P P P N N N N N N N N N N N N N N N N N N N N N N N N N N N N P N RESULTS Immunohistochemical Staining of MT and Panel of Biomarkers Nuclear MT immunopositivity was observed in all the GIST sections, whereas 11 out of 15 (73.3%) GISTs demonstrated immunostaining in the cytoplasm (Table 1). The percentage of nuclear MT-immunopositive cells ranged from 5 to 50%. Most GIST tissue sections (93.3%) were predominantly mildly stained (Fig. 1B), with only 1 tissue section (6.7%) having moderate staining. The negative control for the immunohistochemical staining is shown in Fig. 1A. The IRS scores ranged from 5 to 55. For gastric carcinoma cases, both nuclear and cytoplasmic MT immunopositivity were present in 37 out of 38 (97.4%) tissues. The intensity of immunohistochemical staining among the MT positive gastric carcinoma tissues was variable. There was no MT immunostaining observed in 1 tissue section (2.6%), whereas 24 cases (63.2%) exhibited predominantly mild nuclear staining, 7 cases (18.4%) moderate nuclear staining, and 6 cases (15.8%) strong nuclear staining (Fig. 1C). Nuclear MT expression was signiﬁcantly higher in gastric carcinoma tissue samples when compared with GIST tissue samples when analyzed by percentage stained and IRS (Table 2). With regard to cytoplasmic staining, 4 GISTs (26.7%) had no MT immunostaining and 1 out of the 38 gastric carcinoma cases (2.6%) stained negatively for MT. There was also variable cytoplasmic MT staining in GISTs (Table 1) and gastric carcinomas, but no signiﬁcant difference was found between the two groups. Among the GISTs, 73.3% were positive for CD117 and 86.7% positive for CD34 with 100% staining negatively for S-100 and 93.3% for desmin (Table 1). All the GIST sections were positive for PCNA staining. Statistical Analysis Functional MT-1 and MT-2 MT mRNA Expression in MKN28 Gastric Cancer Cells The GraphPad Prism 5 (GraphPad Software, USA) was used for statistical analyses. The Student t-test was performed for comparison of data between gastric cancer and GIST. Results with P < 0.05 were considered statistically signiﬁcant. MT protein expression was observed in the MKN28 gastric cancer cells (Fig. 2A). As MT has 10 known functional isoforms, an in vitro screening of the expression of the 7 functional MT-1 and 1 MT-2 genes were analyzed in gastric cancer cells to determine which MT gene 270 SOO ET AL. Fig. 1. Immunostaining of MT in GIST and gastric carcinoma tissue samples. (A) Negative control for MT immunostaining (with the addition of diluent in place of primary antibody) in gastric tissue section. Magniﬁcation 200. (B) GIST section displaying predominantly mild nuclear MT staining and cytoplasmic staining of tumor cells. Magniﬁcation 200. (C) Gastric carcinoma section showing strong MT nuclear staining (##) with scattered nuclei being moderately immunostained (#). Magniﬁcation 400. TABLE 2. Nuclear MT expression in GIST and gastric cancer tissue samples DISCUSSION Parameters Nuclear % stained Mean SEM P Value Nuclear IRS Mean SEM P Value GIST (N ¼ 15) Gastric Cancer (N ¼ 38) 16.00 3.44 53.68 5.26 <0.0001 21.09 4.13 91.58 10.64 0.0002 should be used for analysis of the GIST and gastric carcinoma tissues. The MT-3 and MT-4 genes were not evaluated as MT-3 is known to be speciﬁc for the brain and MT-4 present in stratiﬁed epithelium (Cherian et al., 2003). The MT-1A, 1B, 1E, 1G, and 1H transcripts were not detectable in MKN28 gastric cancer cells. As shown in Fig. 2B, MT-1F, 1X, and 2A isoforms were detectable in MKN28 cells by RT-PCR with the MT-2A isoform being the most abundant MT isoform. The speciﬁcity of the MT-1F, 1X, and 2A primers was validated using melting curve analysis (not shown) and demonstrated as single band amplicons in 2% agarose gel electrophoresis after RT-PCR ampliﬁcation (Fig. 2C). MT-2A mRNA Expression The MT-2A gene was selected for further analysis, and MT-2A mRNA expression was determined in 6 GISTs and 6 early stage (Stage 1 and Stage 2) gastric carcinomas selected from the TissueScan Gastroesophageal Tissue qPCR Panel I. The cDNA samples were prenormalized by the manufacturer, and only the MT-2A gene was probed in this experiment. As shown in Fig. 3, there was a signiﬁcant difference in CT values of MT-2A mRNA obtained from patients with GIST when compared with early stage gastric carcinoma (P ¼ 0.0013). As higher CT values are correlated with lower MT-2A gene expression, tissues from patients diagnosed with GIST had a lower MT-2A mRNA expression compared with tissues from gastric carcinoma patients. MTs are known to protect against apoptosis, oxidative stress, proliferation, angiogenesis, and much research has been dedicated to explore their role in tumor progression and other related areas (Cherian et al., 2003; Pedersen et al., 2009). MT has been reported to be overexpressed in several human carcinomas including the breast, ovary, laryngeal, oral cavity, lung (non-smallcell), skin, uterus, and pancreas, but downregulated in other cancers such as gastric, colorectal, liver, and central nervous system tumors (Pedersen et al., 2009). In this study, we observed nuclear expression of MT as determined by immunohistochemistry in all the GISTs, which had mainly positive immunoreactivity for CD117 although the percentage was lower than expected (Ponsaing et al., 2007), positive CD34 staining and negative staining for S-100 protein and desmin. As MTs are known to affect cell proliferation, there is a possibility that MT may be involved in proliferation in GISTs, since PCNA (a proliferative marker) was observed to be immunopositive in all the GIST tissues examined. Some investigators have also argued that nuclear expression of MTs is related to their ability to protect cells against genotoxicity, giving rise to acquisition of malignant phenotype (Dutsch-Wicherek et al., 2008; Pedersen et al., 2009). However, MT expression was considerably lower in GISTs when compared with early stage gastric carcinoma tissue samples. Interestingly, results from both methods used for quantitation of the MT-immunostaining, namely, percentage stained and IRS were convergent and showed signiﬁcantly decreased MT expression in GISTs. We also found that MT-2A mRNA expression was signiﬁcantly downregulated in GISTs compared to gastric carcinoma samples. With regard to MT staining in GIST, Perez-Gutierrez et al. (2007) previously compared MT immunohistochemical expression in 92 GIST and 14 gastrointestinal leiomyosarcomas (GILMS) but found no signiﬁcant difference in MT expression. They also did not ﬁnd any association between MT expression and the anatomical location of the GISTs (gastric when compared with intestinal). GISTs are most commonly found in the stomach, but cases have also been found in the small intestines, large bowel, and oesophagus (Miettinen et al., 1998). MT EXPRESSION IN GISTS AND GASTRIC CANCER 271 Fig. 3. MT-2A mRNA expression in GIST and gastric carcinoma samples. Lower CT value denotes higher MT-2A expression. The values are expressed as CT values as the cDNAs present in the 48 wells have been prenormalized by the manufacturer using the housekeeping gene, b-actin. Values are means of 6 samples for GIST and 6 samples for gastric carcinoma. Error bar ¼ SD. Fig. 2. Expression of MT in MKN28 gastric cancer cells in vitro. (A) Positive MT protein expression in MKN28 cells. Magniﬁcation 200. Bar ¼ 100 lm. (B) MT-1F, 1X, 2A mRNA expression in MKN28 gastric cancer cells. The housekeeping gene G3PDH was used for normalization. A higher DCT value indicates lower expression. Values are means of triplicates. Error bar ¼ SEM. (C) Gel electrophoresis of RT-PCR products of MT-2A and G3PDH. The MT-2A bands (left) and G3PDH bands (right) visualized were obtained from three different samples. The reports on MT expression in gastric cancer have not been consistent. In an immunohistochemical analysis of 112 surgical gastric samples comprising 38 early gastric carcinomas and 74 advanced cases, Tuccari and colleagues showed that MT immunostaining was signiﬁcantly lower in advanced gastric carcinoma cases, along with a higher percentage of MT immunostaining observed in gastric mucosa adjacent to the tumors as compared to the cancer tissues (Tuccari et al., 2000). Similarly, Jansen et al. observed that gastric carcinomas, colorectal adenomas, and carcinomas had signiﬁcantly lower MT-expression as determined by radioimmunoassay than that of corresponding normal-appearing tissues (Janssen et al., 2000). However, using differential display mRNA analysis, Ebert et al. demonstrated overexpression of MT-2A by Northern blot analysis and observed overexpression of MT by immunohistochemistry in tissues obtained from gastric carcinoma (Ebert et al., 2000). Amongst others, Pedersen et al. (2009) have attributed variations observed in MT expression to lack of standardization in the collection of tumors with regard to variability of the tumor stages and MT staining pattern (such as nuclear or cytoplasmic) used in the experimental protocol. The current thinking is that GISTs, which are tumors of mesodermal origin, are derived from the interstitial cells of Cajal (ICC) since the majority of GISTs express CD117 which is similar to that observed in ICCs (Kindblom et al., 1998; Min, 2010). The ICCs were ﬁrst described by Santiago Ramon y Cajal (1893) and have been regarded as pacemaker cells of the gastrointestinal tract (Thuneberg, 1982). Recently, Min (2010) has suggested from ultrastructural observations that GISTs develop from gut stem cells, which could differentiate further to ICC or muscle cells under the control of c-KIT (Torihashi et al., 1999). On the other hand, gastric carcinomas are epithelial tumors of endodermal origin. Differential MT expression in GISTs and gastric carcinoma could, therefore, be due to the histogenesis of the tumors. Moreover, MT isoforms are known to be expressed in a tissue speciﬁc pattern and believed to play distinct roles in different types of cancer cells (Cherian et al., 2003). In summary, we have demonstrated that MT-2A mRNA expression and MT protein expression as detected by 272 SOO ET AL. immunohistochemistry were signiﬁcantly lower in GIST compared with gastric carcinoma. It would appear that nuclear expression of MT may reﬂect an underlying pathobiology of GIST that is distinct from gastric carcinoma. ACKNOWLEDGMENTS The authors thank Dr. Aye Aye Thike from the Department of Pathology, Singapore General Hospital for her assistance in the immunoscoring process, Dr. Manuel Salto-Telez from the Department of Pathology, National University Hospital for facilitating the GIST and gastric cancer TMAs used in this study, and Ms Bay Song Lin for technical assistance. LITERATURE CITED Boku N. 2010. Perspectives for personalization in chemotherapy of advanced gastric cancer. Discov Med 9:84–89. Cajal SR. 1893. Sur les ganglions et plexus nerveux de l’intestin. LR Soc Biol (Paris) 45:217–223. Cherian MG, Jayasurya A, Bay BH. 2003. Metallothioneins in human tumors and potential roles in carcinogenesis. Mutat Res 533:201–209. Coyle P, Philcox JC, Carey LC, Rofe AM. 2002. Metallothionein: the multi-purpose protein. Cell Mol Life Sci 59:627–647. Dutsch-Wicherek M, Sikora J, Tomaszewska R. 2008. The possible biological role of metallothionein in apoptosis. Front Biosci 13: 4029–4038. Ebert MP, Günther T, Hoffmann J, Yu J, Miehlke S, Schulz HU, Roessner A, Korc M, Malfertheiner P. 2000. Expression of metallothionein II in intestinal metaplasia, dysplasia, and gastric cancer. Cancer Res 60:1995–2001. Fock KM, Ang TL. 2010. Epidemiology of Helicobacter pylori infection and gastric cancer in Asia. J Gastroenterol Hepatol 25:479–486. Heinrich MC, Corless CL, Duensing A, McGreevey L, Chen CJ, Joseph N, Singer S, Grifﬁth DJ, Haley A, Town A, Demetri GD, Fletcher CD, Fletcher JA. 2003. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 299:708–710. Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, Ishiguro S, Kawano K, Hanada M, Kurata A, Takeda M, Muhammad Tunio G, Matsuzawa Y, Kanakura Y, Shinomura Y, Kitamura Y. 1998. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279:577–580. Janssen AM, van Duijn W, Oostendorp-Van De Ruit MM, Kruidenier L, Bosman CB, Grifﬁoen G, Lamers CB, van Krieken JH, van De Velde CJ, Verspaget HW. 2000. Metallothionein in human gastrointestinal cancer. J Pathol 192:293–300. Kindblom LG, Remotti HE, Aldenborg F, Meis-Kindblom JM. 1998. Gastrointestinal pacemaker cell tumor (GIPACT): gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal. Am J Pathol 152:1259–1269. Klaassen CD, Liu J, Choudhuri S. 1999. Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol 39:267–294. Liegl-Atzwanger B, Fletcher JA, Fletcher CD. 2010. Gastrointestinal stromal tumors. Virchows Arch 456:111–127. Margoshes M, Vallee B. 1957. A cadmium protein from equine kidney cortex. J Am Chem Soc 79:4813–4814. Mididoddi S, McGuirt JP, Sens MA, Todd JH, Sens DA. 1996. Isoform-speciﬁc expression of metallothionein mRNA in the developing and adult human kidney. Toxicol Lett 85:17–27. Miettinen M, Lasota J. 2006. Gastrointestinal stromal tumors: review on morphology, molecular pathology, prognosis, and differential diagnosis. Arch Pathol Lab Med 130:1466–1478. Miettinen M, Makhlouf H, Sobin LH, Lasota J. 2006. Gastrointestinal stromal tumors of the jejunum and ileum: a clinicopathologic, immunohistochemical, and molecular genetic study of 906 cases before imatinib with long-term follow-up. Am J Surg Pathol 30:477–489. Miettinen M, Sarlomo-Rikala M, Kovatich AJ, Lasota J. 1999b. Calponin and h-caldesmon in soft tissue tumors: consistent h-caldesmon immunoreactivity in gastrointestinal stromal tumors indicates traits of smooth muscle differentiation. Mod Pathol 12:756–762. Miettinen M, Sarlomo-Rikala M, Lasota J. 1998. Gastrointestinal stromal tumours. Ann Chir Gynaecol 87:278–281. Miettinen M, Sarlomo-Rikala M, Lasota J. 1999a. Gastrointestinal stromal tumors: recent advances in understanding of their biology. Hum Pathol 30:1213–1220. Miettinen M, Sobin LH, Sarlomo-Rikala M. 2000. Immunohistochemical spectrum of GISTs at different sites and their differential diagnosis with a reference to CD117 (KIT). Mod Pathol 13:1134–1142. Min KW. 2010. Gastrointestinal stromal tumor: an ultrastructural investigation on regional differences with considerations on their histogenesis. Ultrastruct Pathol 34:174–188. Panani AD. 2008. Cytogenetic and molecular aspects of gastric cancer: clinical implications. Cancer Lett 266:99–115. Parkin DM, Bray F, Ferlay J, Pisani P. 2005. Global cancer statistics, 2002. CA Cancer J Clin 55:74–108. Pedersen M, Larsen A, Stoltenberg M, Penkowa M. 2009. The role of metallothionein in oncogenesis and cancer prognosis. Prog Histochem Cytochem 44:29–64. Perez-Gutierrez S, Gonzalez-Campora R, Amerigo-Navarro J, BeatoMoreno A, Sanchez-Leon M, Pareja Megia JM, Virizuela-Echaburu JA, Lopez-Beltran A. 2007. Expression of P-glycoprotein and metallothionein in gastrointestinal stromal tumor and leiomyosarcomas. Clinical implications. Pathol Oncol Res 13:203–208. Ponsaing LG, Kiss K, Hansen MB. 2007. Classiﬁcation of submucosal tumors in the gastrointestinal tract. World J Gastroenterol 13:3311–3315. Rossi F, Ehlers I, Agosti V, Socci ND, Viale A, Sommer G, Yozgat Y, Manova K, Antonescu CR, Besmer P. 2006. Oncogenic Kit signaling and therapeutic intervention in a mouse model of gastrointestinal stromal tumor. Proc Natl Acad Sci USA 103:12843–12848. Sarlomo-Rikala M, Kovatich AJ, Barusevicius A, Miettinen M. 1998. CD117: a sensitive marker for gastrointestinal stromal tumors that is more speciﬁc than CD34. Mod Pathol 11:728–734. Thirumoorthy N, Manisenthil Kumar KT, Shyam Sundar A, Panayappan L, Chatterjee M. 2007. Metallothienein: an overview. World J Gastroenterol 13:993–996. Thuneberg L. 1982. Interstitial cells of Cajal: intestinal pacemaker cells? Adv Anat Embryol Cell Biol 71:1–130. Torihashi S, Nishi K, Tokutomi Y, Nishi T, Ward S, Sanders KM. 1999. Blockade of kit signaling induces transdifferentiation of interstitial cells of cajal to a smooth muscle phenotype. Gastroenterology 117:140–148. Tuccari G, Giuffre G, Arena F, Barresi G. 2000. Immunohistochemical detection of metallothionein in carcinomatous and normal human gastric mucosa. Histol Histopathol 15:1035–1041.