Experimental and Molecular Pathology 72, 172–177 (2002) doi:10.1006/exmp.2001.2419, available online at http://www.idealibrary.com on
Gastrointestinal Stromal Tumors: Are They of Cajal Cell Origin?
Xiaojuan Wang,* Ichiro Mori,* Weihua Tang,* Hirotoshi Utsunomiya,* Misa Nakamura,* Yasushi Nakamura,* Genying Zhou,† Kennichi Kakudo* *Department of Pathology, Wakayama Medical University, 811-1 Kimiidera, Wakayama City, Japan 641-0012; and †Department of Pathology, West Campus, Shandong University, 44 West Wenhua Road, Jinan, China 250012
Received September 20, 2001
Recently some reports have suggested that gastrointestinal stromal tumors (GIST) might originate from the interstitial cells of Cajal or differentiate into them because they express c-kit and/or CD34 and indicated that the majority of previously diagnosed smooth muscle tumors (SMT) actually belong to GIST, but are not true SMT. We, therefore, detected c-kit, CD34, SMA, and S-100 in 106 Chinese cases of gastrointestinal tumors, which were histopathologically diagnosed as smooth muscle tumors originally, to demonstrate the immunophenotypes of these tumors. The results showed that 73 cases had immunoreaction with c-kit and/or CD34, of which 48 cases showed coexpression with either SMA or S-100 or with both. A correlation between the immunophenotypes and known histopathological parameters was also shown here based on follow-up data. We suggest that the concept of GIST should not be used as an umbrella to cover all gastrointestinal mesenchymal tumors, but be defined in a narrow term as differing from true smooth muscle tumors. 䉷 2002 Elsevier Science (USA) Key Words: Cajal cell; GIST; immunohistochemistry; alimentary tract; c-kit; CD34.
or schwannoma traditionally. The term gastrointestinal stromal tumor (GIST) was introduced by Mazur and Clark in 1983 as a histogenetically neutral term referring to the main group of mesenchymal tumors of the gastrointestinal tract, which could be verified neither from smooth muscle origin nor neurogenic origin (Masur and Clark, 1983). GIST has also been designated as an umbrella term to cover all mesenchymal tumors whether leiomyomas, schwannomas, or neither (Rosai, 1996). Recently some investigators have suggested that GIST might originate from the interstitial cells of Cajal or differentiate into them because they express ckit and/or CD34 (Hirota, 1998; Kindblom, 1998; SarlomaRikala, 1998; Sircar, 1999; Kluppel, 1998) and indicated that the majority of previously diagnosed smooth muscle tumors (SMT) actually belong to GIST but are not true SMT. We therefore immunohistochemically detected c-kit, CD34, SMA, and S-100 in 106 Chinese cases of gastrointestinal tumors, which were originally diagnosed as smooth muscle tumors histopathologically, to demonstrate the immunophenotypes of these tumors. A correlation between the immunophenotypes and known histopathological parameters, such as mitotic index, tumor size, cellularity, tumor location, tumor necrosis, tumor hemorrhage, and invasive growth, was also shown.
The primary mesenchymal cell tumors of the gastrointestinal tract were uniformly classified as smooth muscle tumors (leiomyoma, leiomyoblastoma, or leiomyosarcoma)
0014-4800/02 $35.00 䉷 2002 Elsevier Science (USA) All rights reserved.
IS GIST A CAJAL CELL ORIGIN?
MATERIALS AND METHODS
Patients and tumors. One hundred and six cases were obtained from the Department of Pathology, the Affiliated Hospital of Shandong Medical University, Jinan, China, in the 30-year period from 1969 to 1999. All of these surgical specimens were routinely fixed in Formalin and embedded in paraffin. The original diagnoses included leiomyoma, leiomyoblastoma, and leiomyosarcoma. The cases occurred between the ages of 6 days and 73 years, with a mean of 49.3, and the tumors ranged in size from 0.5 to 30 cm. The male to female ratio was 1.4:1. Twenty-five cases were located in the esophagus, 37 in the stomach, 33 in the small intestine, and 11 in the large intestine. At least four to five blocks were available for evaluation in each case. Follow-up data were available only in 65 cases and the follow-up period ranged from 12 months to 31 years (average 8.6 years). Patients who developed a metastasis or those died of the tumor were classified as malignant cases. Tumors with invasive growth in the peripheral tissue microscopically were also diagnosed as malignant. The other cases without the evidence of malignancy were classified as benign in this study. There is still a slight chance of malignancy in our benign group. In addition, a no-mitosis group and a mitosis group were classified, based on the mitotic number as previously described (Evans, 1985; McGrath, 1987; Xiaojuan, 1994). Briefly, 10 high-power fields in one group were observed to find a mitotic figure at the fields of cellularity; a total of five groups were observed in each case. Simultaneously (1) histological type, i.e., leiomyoma type, schwannoma type, leiomyoblastoma type, and mixed type; (2) cellularity, i.e., low, intermediate, and high; (3) tumor size, checked in surgical and pathological records; (4) tumor location; (5) tumor
TABLE 1 Dilution and Sources of the Antibodies in the Study Antibody c-kit CD34 SMA S-100 Ki-67* p53
Clone Poly Qbend10 1 A4 Poly MIB-1 DO7
Pretreatment Microwave Microwave None None None Microwave
1:20 1:25 1:400 1:500 1:50 1:400
1 2 3 4 5 4
Note. (1) Immuno-Biochemical Laboratories, Fujioka, Japan. (2) Novocastra, New Castle, UK. (3) Sigma Chemical, St. Louis, MO. (4) DAKO, Kyoto, Japan. (5) Immunotech, Marseille Cedex, France. a The results of immunostaining for Ki-67 and p53 were reported in another journal.
FIG. 1. (A) The tumor shown is composed of spindle cells with eosinophilic cytoplasma, low cellularity, and few mitotic figures and was diagnosed as a leiomyoma previously (HE; original magnification, x200). (B) The figure shows a tumor composed of short spindle cells, arranged in a solid sheet with high cellularity and increased mitotic figures, which was diagnosed as a leiomyosarcoma previously (HE; original magnification, x200).
necrosis; and (6) tumor hemorrhage were also noted in each case. Immunohistochemistry. A standard labeled streptavidin– biotin (LSAB) method was performed. Antibody dilution, sources, and pretreatment of the primary antibodies are shown in Table 1. Positive and negative controls were used simultaneously. The results were evaluated by the following criteria: No staining, faint staining, and distinct staining of fewer than 10% of tumor cells were considered negative and distinct staining of more than 10% of tumor cells was judged positive. Statistical analysis. Time-independent continuous variables were evaluated using the Student’s t test and the 2 test was used for categorical data. Survival analysis was done with StatView-J 5.0 statistical software packages (SAS Institute
WANG ET AL.
TABLE 2 Immunohistochemical Results with Clinicohistological Findings in the Study
Group Benign Malignant Location Esophagus Stomach Small intestine Large intestine Tumor size Ɒ5 cm ⬍5 cm Histological type Leiomyoma Schwannoma Epithelioid Mixed Cellularity Low Middle High Mitotic index Ɒ1/10 HPF ⬍1/10 HPF Hemorrhage With Without Necrosis With Without Invasive growth With Without Ki-67 ⫹ ⫺ p53 ⫹ ⫺ Follow-upb Alive Dead a b
SMA ⫹ S-100
13 34 22 4
11 1 2 2
1 2 4 1
0 0 2 2
0 0 3 2
0.0355 (⫽ 0.0007)a ⬍0.0001 (⫽ 0.0001)
51 7 7 8
15 1 0 0
6 1 1 0
4 0 0 0
3 1 1 0
30 29 14
14 2 0
6 1 1
2 1 1
3 1 1
0.0245 (⫽ 0.0040)
0.0276 (⫽ 0.0053)
0.0025 (⫽ 0.0003) 0.0011
c-kit/CD34 vs SMA. Follow-up data were available only in 65 cases.
Inc., Cary, NC). For univariate analysis of time-dependent variables, the Kaplan–Meier method and the log rank test were used to determine significant prognostic factors. In multivariate analysis, a Cox proportional hazards regression model was used for all factors found to be significant in univariate analysis. A P value less than 0.05 was considered statistically significant.
RESULTS Microscopic features. Microscopically, the tumors were mainly composed of spindle cells with eosinophilic cytoplasma, e.g., leiomyoma type (79 cases, 74.5%), a few tumors showed a predominantly epithelioid pattern, e.g., leiomyoblastoma (8 cases, 7.5%), a palisading nuclear pattern
IS GIST A CAJAL CELL ORIGIN? TABLE 3 Detailed Results of the Coexpression of c-kit, CD34, SMA, and S-100 in the Study Antibodies c-kit(⫹) CD34(⫹)/36 c-kit⫹, CD34⫺, SMA⫺, S-100⫺ ⫹SMA ⫹S-100 ⫹SMA, ⫹S-100 c-kit(⫹) CD34(⫺)/25 c-kit⫹, CD34⫺, SMA⫺, S-100⫺ ⫹SMA ⫹S-100 ⫹SMA, ⫹S-100 c-kit(⫺) CD34(⫹)/12 c-kit⫺, SMA⫺, S-100⫺ ⫹SMA ⫹S-100 ⫹SMA, ⫹S-100
multivariate analysis, only Ki-67 (P ⫽ 0.0093) and cellularity (P ⫽ 0.0040) were significant by independent prognostic factors.
17 15 3 1 5 9 4 7 3 3 3 3
similar to nerve sheath tumors, as in the schwannoma type (10 cases, 9%), and some tumors showed an admixture of variants with storiform arrangement, solid sheets, or myxoid change, as the mixed type (9 cases, 8.4%). (Figs. 1a and 1b) Immunohistochemical findings with the results of the follow-up. Of the 106 cases, 73 cases (68.86%) showed expression of c-kit and/or CD34, 16 were positive for SMA alone, 8 for S-100 alone, 4 for both SMA and S-100, and 5 cases were negative for all the four markers. The clinical and microscopic findings and detailed immunohistochemical results are summarized in Tables 2 and 3. Of the 73 cases positive for c-kit and/or CD34, 36 were positive for both ckit and CD34; of those, 17 cases were positive for both ckit and CD34, 15 cases were positive for SMA, 3 cases for S-100, and 1 case for both SMA and S-100 (Figs. 2a–2d). Positive for c-kit alone were 25 cases; of them, 5 were positive only cases for c-kit, 9 cases for SMA, 4 cases for S-100, and 7 cases for both SMA and S-100. Positive for CD34 alone were 12 cases; of those, 3 cases were positive only for CD34, 3 cases for SMA, 3 cases for S-100, and 3 cases for both SMA and S-100. Survival analysis. Univariate analysis demonstrated that invasive growth (P ⫽ 0.0021), cellularity (P ⫽ 0.0001), hemorrhage (P ⫽ 0.0001), tumor necrosis (P ⫽ 0.0004), Ki-67 index (P ⫽ 0.0002), and p53 immunostaining (P ⫽ 0.0021) correlated significantly with a poor prognosis. However, no such correlation was observed between the expression of c-kit and/or CD34 with the prognosis. On
DISCUSSION The interstitial cells of Cajal (ICC), the pacemaker cells, form a complex network intercalated between the autonomic nerves and the muscle walls of the gastrointestinal tract and c-kit receptor is a sensitive marker for ICC. Recently, Hirota (1998) and Kindblom et al. (1998) suggested that GIST might originate from the ICC or differentiate toward Cajal cells because of the positive expression of c-kit and/or CD34. The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor protein that binds to stem cell factor. An interaction between c-kit protein and stem cell factor is essential for the normal development of melanocytes, erythrocytes, germ cells, mast cells, and ICC in the gastrointestinal tract (Tssura, 1994; Torihashi, 1995; Ward, 1994; Huizinga, 1995). CD34 antigen is an 11-kDa glycosylated transmembrane protein, originally described as a hematopoietic stem cell marker, that has been observed in a wide range of normal tissue and tumors, as well as in GIST and normal ICC (Monihan, 1994; van de Rijn, 1994; Miettinen, 1995). Our results in the present study reconfirmed that a majority of the tumors previously diagnosed as smooth muscle tumors were actually GIST, which had immunoreaction against ckit and/or CD34. Indeed, in the gastrointestinal tract, a true smooth muscle tumor was not frequent, especially in the stomach and intestine, and most of them were located in the esophagus had benign behavior (van de Rijn, 1994; Miettinen, 1995). But, on the other hand, of those 73 cases positive for c-kit and/or CD34, only 25 cases were positive for c-kit and/or CD34 alone. The other 48 cases showed coexpression with SMA, S-100, or with both SMA and S-100. These results were in good agreement with Newman’s suggestion that GIST could be divided into various different subtypes, such as myogenic, neurogenic, biphasic, and undifferentiated types, because of their positive expression not only for c-kit and/or CD34 but also for myogenic or neurogenic markers (Newman, 1991). A majority of those tumors positive for c-kit and/ or CD34 (so-called GIST) demonstrated double or multiple differentiations immunohistochemically (Hurlimann, 1991; Tazawa, 1999). Together with our results, this suggested that a GIST might be considered a tumor originating from immature mesenchymal cells (precursor cells) and exhibits differentiation toward many different cell types, including the ICC and
WANG ET AL.
FIG. 2. Immunohistochemical staining shows tumor cells positive for c-kit (A), CD4 (B), SMA (C), and S-100 (D) (LSAB; original magnification, x200).
myogenic and neurogenic cells, or multiple differentiations. Therefore, gastrointestinal mesenchymal tumors might be divided into GIST (a narrow definition, a tumor positive for ckit and/or CD34, regardless of whether it shows coexpression with SMA, S-100, or both), smooth muscle tumors (positive for SMA alone), schwannoma (positive for S-100 alone), and the others, in the light of immunostaining. GIST should not be used as an umbrella term to cover all mesenchymal tumors of the gastrointestinal tract because a true smooth muscle tumor often shows benign behavior (van de Rijn, 1994; Miettinen, 1995; Emory, 1999). In our practice, it seemed relatively difficult to distinguish GIST from the true smooth muscle tumor by using the routine light method. Similar views were also noted in other related
literature (Miettinen, 2001; Schmid, 2000; Sakurai, 1998). The histological pattern of a GIST appears similar to that of the true smooth muscle tumor or schwannoma and it is not easy to prove the origin of the different tumor cells with HE staining and light morphology. Thus, immunostaining might play an important role in distinguishing different mesenchymal tumors in the gastrointestinal tract. So far, the criteria to predict the biological behavior of GIST remain vague and there is no reliable cutoff between benign and malignant cases. We referred to the criterion of mitotic index used in smooth muscle tumors of the gastrointestinal tract before in which the results demonstrated that there was a significant difference between the mitotic index and the biological behavior of GIST and that no deceased case was
IS GIST A CAJAL CELL ORIGIN?
found in our no-mitosis group, except for three cases with a mitotic index of 1/10 HPF dying of recurrence and metastasis of the tumors. According to the criterion, the other histological parameters also showed a statistically significant difference between benign and malignant lesions. Compared with the other histological parameters, mitotic index seems a relatively simple and reproducible method and the method might improve our ability to predict the malignant potential in patients with GIST. In conclusion, we suggested that the concept of GIST should be used as a narrow definition separating them from the true smooth muscle tumor and true neurogenic tumor. Mitotic index might be considered a helpful criterion to predict the biological behavior of patients with GIST.
We thank Emiko Taniguchi, Tomoko Kagiya, and Misako Sato for preparing histological sections. This work was supported by Grant in Aid for scientific research (B), Japan, No. 10045073.
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