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Cellular Origin of Gastrointestinal Stromal Tumors.

A Study of 27 Cases

Gastrointestinal stromal tumors (GISTs) are probably one of the most controversial gastrointestinal tumors in regard to cell origin, differentiation, nomenclature, and prognosis.[1] Historically, Stout[2] assumed that GISTs were derived from smooth muscle based on their morphologic resemblance to smooth muscle tumors in other anatomic sites. Appelman and Helwig have postulated that gastric stromal tumor may originate from mesenchymal cells other than smooth muscle cells.[3-5] The spindle cell neoplasms of the gastrointestinal tract were called leiomyoma or leiomyosarcoma, and the predominant round or polygonal cell variant was named epithelioid leiomyoma, leiomyoblastoma, or epithelioid leiomyosarcoma.[3-9] Despite advanced immunohistochemical and ultrastructure studies, the line of differentiation (smooth muscle, neural, gastrointestinal, autonomic nerve, or undifferentiated) is still inconclusive.[10-15] As a result, the noncommittal term gastrointestinal stromal tumor has been adopted to describe this heterogeneous group of neoplasms.[16]

Recently, Kindblom et al and others have proposed that most stromal tumors originate from a mesenchymal stem cell that differentiates toward an interstitial cell of Cajal (ICC) phenotype.[17-20] Also known as pacemaker cells, ICCs are a population of cells in the gastrointestinal tract that play a role in the control of gut motility.[21] The ICCs express the c-kit (CD117) proto-oncogene encoding a type III tyrosine kinase (KIT) receptor, a ligand known as stem cell factor (SCF).[22] Maturation of ICC depends on SCF-KIT interaction.[23] Evidence now suggests that CD34-positive stromal tumor, known to be c-kit positive, may differentiate into ICC-like cells.[17,22]

The purpose of the present investigation is to test the hypothesis that GISTs originate from CD34-positive stem cells and differentiate toward an ICC phenotype by immunohistochemical studies using selected lineage-directed monoclonal antibodies for ICCs (c-kit), smooth muscle (desmin), Schwann cells (S100 protein), stem cells (CD34), myofibroblasts ([Alpha]-actin), and proliferative cells (MIB-1).

MATERIALS AND METHODS

Twenty-seven cases of GISTs were collected for 14 years (1985-1999), including 8 benign (leiomyoma), 15 malignant primary (leiomyosarcoma), and 4 metastatic to the liver. According to Appelman's criterion, we categorized these 27 cases as malignant ([is greater than] 5 mitoses per 50 high-power fields), borderline ([is less than] 5 mitoses per 50 high-power fields; [is greater than] 5 cm in size), and benign ([is less than] 5 mitoses per 50 high-power fields; [is less than] 5 cm). The patients ranged in age from 35 to 78 years (mean, 53 years; median, 53 years). The female-male ratio was 15:12. The GISTs were from the stomach (11), small bowel (9), cecum (1), rectum (1), abdominal nodule (1), and metastatic to the liver (4) (Table 1).

Table 1. Clinic Data of 27 Gastrointestinal Stromal Tumors
Parameters Characteristics

Age, y 35-78
 (mean, 53; median, 53)

Sex
 Male 12
 Female 15

Primary tumors
 Gastric 11
 Small bowel 9
 cecum 1
 Rectum 1
 Abdominal nodule 1
 Metastatic tumors to liver 4
Total 27


Specimens were fixed in 10% buffered formalin, embedded in paraffin tissue, and stained with hematoxylin-eosin using standard procedures. Sections from each case were studied by a modified avidin-biotin complex immunoperoxidase method using a panel of antibodies (Table 2).
Table 2. Antibody Panel

Antibody Clone Source Dilution

C-kit (CD117) C-19 Cruz Biotechnology 1:40
CD34 QBEnd/10 Novocastra 1:25
Vimentin V9 Zymed 1:50
Desmin ZC18 Zymed 1:50
[Alpha]-Actin 1A4 Sigma 1:200
S100 ... Dako 1:1000
MIB-1 7B11 Zymed 1:200


RESULTS

The gross features of GISTs usually included a granular, irregular surface with frequent areas of hemorrhage, necrosis, and cystic change (Figure 1). They lacked the characteristic gross appearance of smooth muscle tumors of the uterus in that they did not have a bulging, whorled cut surface. They were commonly well circumscribed, lobulated, or multilobulated and were either partly or predominantly extramural in location. Most tumors were centered in the submucosa, muscularis propria, or both. Both benign and malignant tumors showed ulceration of the overlying mucosa. Therefore, benign and malignant tumors had overlapping gross features and were indistinguishable by gross examination alone except for size.

[Figure 1 ILLUSTRATION OMITTED]

Microscopically, the benign GISTs (leiomyoma) were composed of spindle cells with pale or eosinophilic fibrillar cytoplasm. The cells varied little in size and shape and were typically arranged in either whorls or long fascicles. Their nuclei were uniform with either low or zero mitotic count. Sometimes, perinuclear vacuoles that indented the nuclei at one pole were identified (Figure 2).

[Figure 2 ILLUSTRATION OMITTED]

In contrast, malignant or metastatic GISTs (leiomyosarcoma) consisted of high cellularity with an increased nuclear-cytoplasmic ratio. Bizarre cells, nuclear pleomorphism, and mitotic figures were easily found (Figure 3). The cytoplasm was eosinophilic.

[Figure 3 ILLUSTRATION OMITTED]

Immunoperoxidase stains of normal gastrointestinal tract showed both c-kit and CD34-positive cells surrounding the Auerbach ganglia plexus (Figure 4). Twenty-seven of 27 tumors strongly expressed c-kit (Figure 5). Fourteen of 27 tumors were positive for CD34 (Figure 6). Of the malignant GISTs, 14 of 19 were positive for CD34; of the benign tumors, 0 of 8 were positive for CD34. Thus, CD34 was the best indicator of malignant phenotype (Table 3).

[Figures 4-6 ILLUSTRATION OMITTED]

Table 3. Immunohistochemical Analysis of Gastrointestinal Stromal Tumors (GISTs)(*)
 Patient
No./Sex/Age,
 y Location Diagnosis

 1/M/43 Stomach Gastric stromal tumor
 2/F/51 Stomach Smooth muscle neoplasm
 3/M/43 Stomach Leiomyosarcoma
 4/M/54 Stomach Leiomyosarcoma
 5/M/52 Stomach Leiomyosarcoma
 6/F/62 Stomach Gastric stromal tumor
 7/F/53 Stomach GIST
 8/M/50 Stomach Leiomyosarcoma
 9/F/78 Small intestine GIST, Smooth muscle
 differentiation
 10/M/44 Small intestine Leiomyosarcoma
 11/F/49 Small intestine Malignant stromal tumor
 from leiomyosarcoma
 12/M/60 Small intestine Malignant stromal tumor
 13/F/63 Small intestine Neurofibromatosis
 14/M/63 Duodena Malignant stromal tumor
 from leiomyosarcoma
 15/M/64 Rectum Malignant stromal tumor
 16/F/58 Liver Leiomyosarcoma
 (metastatic)
 17/F/56 Liver Leiomyosarcoma
 (metastatic)
 18/F/53 Liver Leiomyosarcoma
 (metastatic)
 19/M/46 Liver Leiomyosarcoma
 (metastatic)
 20/M/58 Gastrointestinal Leiomyoma
 junction
 21/M/55 Gastrointestinal Leiomyoma
 junction
 22/F/49 Abdominal mass Leiomyoma
 23/F/46 Small intestine Leiomyoma
 24/F/37 Small intestine Stromal tumor
 25/F/35 Cecum GIST
 26/F/54 Stomach GIST
 27/F/61 Small intestine GIST

Diagnosis C-kit CD34 Vimentin

Gastric stromal tumor + F+(*) +
Smooth muscle neoplasm + + F+
Leiomyosarcoma + + +
Leiomyosarcoma + + +
Leiomyosarcoma + + +
Gastric stromal tumor + + +
GIST + + +
Leiomyosarcoma + + +
GIST, Smooth muscle F+ - -
 differentiation
Leiomyosarcoma + - +
Malignant stromal tumor + - F+
 from leiomyosarcoma
Malignant stromal tumor + - +
Neurofibromatosis + + +
Malignant stromal tumor + F+ +
from leiomyosarcoma
Malignant stromal tumor F+ + +
Leiomyosarcoma + + +
 (metastatic)
Leiomyosarcoma + + +
 (metastatic)
Leiomyosarcoma + - +
 (metastatic)
Leiomyosarcoma + + +
 (metastatic)
Leiomyoma + - +
Leiomyoma + - +
Leiomyoma + - +
Leiomyoma + - +
Stromal tumor + - -
GIST + - +
GIST + - +
GIST + - +

Diagnosis [Alpha]-Actin Desmin

Gastric stromal tumor F+ -
Smooth muscle neoplasm F+ -
Leiomyosarcoma - -
Leiomyosarcoma F+ -
Leiomyosarcoma - +
Gastric stromal tumor + -
GIST F+ F+
Leiomyosarcoma - -
GIST, Smooth muscle + +
 differentiation
Leiomyosarcoma + +
Malignant stromal tumor + -
 from leiomyosarcoma
Malignant stromal tumor - -
Neurofibromatosis F+ -
Malignant stromal tumor F+ -
from leiomyosarcoma
Malignant stromal tumor F+ -
Leiomyosarcoma + -
 (metastatic)
Leiomyosarcoma + -
 (metastatic)
Leiomyosarcoma + +
 (metastatic)
Leiomyosarcoma + -
 (metastatic)
Leiomyoma + +
Leiomyoma + +
Leiomyoma + F+
Leiomyoma + +
Stromal tumor + -
GIST F+ -
GIST + -
GIST + -

Diagnosis S100 MIB-1, %

Gastric stromal tumor - 3
Smooth muscle neoplasm - <1
Leiomyosarcoma - -
Leiomyosarcoma + 1
Leiomyosarcoma - -
Gastric stromal tumor - <1
GIST - 3
Leiomyosarcoma - <1
GIST, Smooth muscle - <1
 differentiation
Leiomyosarcoma - -
Malignant stromal tumor - -
 from leiomyosarcoma
Malignant stromal tumor - <1
Neurofibromatosis - <1
Malignant stromal tumor + 5
 from leiomyosarcoma
Malignant stromal tumor - 60
Leiomyosarcoma - -
 (metastatic)
Leiomyosarcoma - 40
 (metastatic)
Leiomyosarcoma - 50
 (metastatic)
Leiomyosarcoma - -
 (metastatic)
Leiomyoma + -
Leiomyoma - -
Leiomyoma - <1
Leiomyoma - -
Stromal tumor + -
GIST F+ <1
GIST - 3
GIST - -


(*) F indicates focal; plus sign, positive; and minus sign, negative

Most GISTs expressed vimentin (25/27) and a-actin (23/27), but desmin and S100 were only positive in 9 and 5 of the 27 tumors, respectively. There was a variable expression of MIB-1 (from 0% to 3% in benign lesions and up to 60% in malignant tumors). All benign GISTs were CD34 negative (0/8), with zero to low expression ([is less than] 3%) of MIB-1. However, some malignant GISTs (5/19) were CD34 negative, 4 of them from small intestine and 1 liver metastasis (Table 4).

Table 4. Immunohistochemical Analysis of 27 Gastrointestinal Stromal Tumors (GISTs)
GISTs C-kit CD34 Vimentin

Benign (8) 8/8 0/8 7/8

Malignant (19)
 Stomach (8) 8/8 8/8 8/8
 Small intestine (5) 5/5 0/5 4/5
 Duodenum (1) 1/1 1/1 1/1
 Rectum (1) 1/1 1/1 1/1
 Metastatic to liver (4) 4/4 3/4 4/4

GISTs [Alpha]-Actin Desmin

Benign (8) 8/8 4/8

Malignant (19)
 Stomach (8) 5/8 2/8
 Small intestine (5) 4/5 2/5
 Duodenum (1) 1/1 0/1
 Rectum (1) 1/1 0/1
 Metastatic to liver (4) 3/4 1/3

GISTs S100 MIB-1, %

Benign (8) 3/8 0-3

Malignant (19)
 Stomach (8) 1/8 0-3
 Small intestine (5) 1/5 0-<1
 Duodenum (1) 0/1 5
 Rectum (1) 0/1 60
 Metastatic to liver (4) 0/3 0-50


COMMENT

This is the first report that all the benign GISTs stained negative for CD34. The results of this study suggest that GISTs originate from CD34-positive stem cells, which differentiate to express the pacemaker cell phenotype. The lack of expression of CD34 in the benign GISTs may indicate that they are composed of more mature ICCs, whereas malignant GISTs are composed of dedifferentiated ICCs and express stem cell markers (CD34).

The ICCs express KIT receptor. The proto-oncogene c-kit was first identified as the cellular homologue of the oncogene v-kit, found in the feline sarcoma virus, which causes multicentric fibrosarcoma in the domestic cat? The proto-oncogene c-kit is a transmembrane tyrosine kinase receptor that belongs to the same family of receptors as those for platelet-derived growth factor and colony-stimulating factor 1. In human beings, the c-kit gene maps to chromosome 4 (4q11-12) in close proximity to the plateletderived growth factor receptor and to the gene for epidermal growth factor. The c-kit gene is expressed primarily on hematopoietic stem cells, mast cells, ICCs, melanocytes, and germ cells.

The ultrastructure features of ICCs are many and include subplasmalemmal actin filament bundles, numerous large mitochondria, abundant smooth endoplasmic reticulum, microtubules, caveolae, interdigitating cytoplasmic processes, incomplete external lamina, and synapselike contracts. Thus, these cells have both myoid and neural features, which would explain prior ultrastructural data regarding the electron microscopic features of GISTs.[11]

Vanderwinden and colleagues[24] clearly demonstrated that CD34 and c-kit immunoreactivity reside in closely adjacent, but not overlapping, cell populations in human intestine by double immunofluorescence studies combined with nuclear counterstain and confocal laser microscopy. They found that CD34+ cells are fibroblasts that express the fibroblast marker prolyl 4-hydroxylase. These cells are adjacent to ICCs but distinct from ICCs. These findings challenge the hypothesis that GISTs originate from ICCs.

However, these findings open the possibility that somatic mutations of c-kit may represent part of the oncogenic process rather than an indication of the origin of ICCs per se. This is supported by the finding that CD117, the c-kit proto-oncogene product, is in fact a more specific marker for GISTs than is CD34.[19] In addition, an in-frame deletion or a point mutation in exon 11 of c-kit occurs preferentially in malignant versus benign GISTs,[25] and c-kit mutation is associated with a poor prognosis in patients with GISTs.[26] There is also a report of familial GISTs with a germline mutation (exon 11 deletion between the transmembrane and tyrosine kinase domain) of the c-kit gene.[27] A report of a woman with leiomyomatosis and a leiomyosarcoma arising from a leiomyoma may indicate c-kit is important in the development and malignant transformation of GISTs.[28] All these may explain why malignant GISTs express both c-kit and CD34 but benign GISTs express c-kit only. This is possibly attributable to gain-of-function mutations of the c-kit gene.[22]

References

[1.] Franquemont DW. Differentiation and risk assessment of gastrointestinal stromal tumors. Am J Clin Pathol. 1995;103:41-47.

[2.] Stout AP. Bizarre smooth muscle tumors of the stomach. Cancer. 1962;15: 400-409.

[3.] Appelman HD, Helwig EB. Gastric epithelioid leiomyoma and leiomyosarcoma (leiomyoblastoma). Cancer. 1976;38:708-728.

[4.] Appelman HD, Helwig EB. Cellular leiomyomas of the stomach in 49 patients. Arch Pathol Lab Med. 1977;101:373-377.

[5.] Appelman HD, Helwig EB. Sarcomas of the stomach. Am J Clin Pathol. 1977;67:2.

[6.] Morrissey K, Cho ES, Gray GF Jr, et al. Muscular tumors of the stomach: clinical and pathological study of 113 cases. Ann Surg. 1973;178:148-155.

[7.] Ranchod M, Kempson RL. Smooth muscle tumors of the gastrointestinal tract and retroperitoneum. Cancer. 1977;39:255-262.

[8.] Shiu MH, Farr GH, Papachristou DN, et al. Myosarcoma of the stomach: nature history, prognostic factors and management. Cancer. 1982;49:177-187.

[9.] Lindsay PC, Ordonez N, Raaf JH. Gastric leiomyosarcoma: clinical and pathological review of fifty patients. J Surg Oncol. 1981 ;18:399-421.

[10.] Herrera GA, Cerezo L, Jones JE, et al. Gastrointestinal autonomic nerve tumors: "plexosarcoma". Arch Pathol Lab Med. 1989;113:846-853.

[11.] Mackay B, Ro J, Floyd C, Ordonez NG. Ultrastructural observations on smooth muscle tumors. Ultrastruct Pathol. 1987;11:593-607.

[12.] Pike AM, Lloyd RV, Appelman HD. Cell markers in gastrointestinal stromal tumors. Hum Pathol. 1988;19:830-834.

[13.] Ma CK, Amin MB, Kintanar E, Linden MD, Zarbo RJ. Immunohistologic characterization of gastrointestinal stromal tumors: a study of 82 cases compared with 11 cases of leiomyoma. Mod Pathol. 1993;6:139-145.

[14.] Van de Riji M, Hendrickson MR, Rouse RV. CD34 expression by gastrointestinal stromal tumors. Hum Pathol. 1994;25:766-771.

[15.] Miettinen 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;19:207-216.

[16.] Silverberg SG, DeLellis RA, Frable WJ. The stomach. In: Principles and Practice of Surgical Pathology and Cytology. Vol 2.3rd ed. New York: Churchill Livingstone; 1997:1699.

[17.] Kindblom LG, Remotti CE, 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: 1259-1269.

[18.] Sircar K, Hewlett B, Riddell RH. Most gastrointestinal stromal tumors arise from interstitial cells of Cajal. Mod Pathol. 1998;11:401A.

[19.] Sarlomo-Rikala M, Kovatich AJ, Barusevivius A, Miettinen M. CD117: a sensitive marker for a gastrointestinal stromal tumor that is more specific than CD34. Mod Pathol. 1998;11:728-734.

[20.] Sakurai S, Fukasawa T, Chong JM, Tanaka A, Fukayama M. Embryonic form of smooth muscle myosin heavy chain (SMemb/MHC-B) in gastrointestinal stromal tumor and interstitial cells of Cajal. Am J Pathol. 1999;154:23-28.

[21.] Hagger R, Finlayson C, Jeffrey L, Kumar D. Role of the interstitial cells of Cajal in the control of gut motility. Br J Surg. 1997;84:445-450.

[22.] Hirota S, Isozaki K, Moriyama Y, et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science. 1998;297:577-580.

[23.] Vliagoftis H, Worobec AS, Metcalfe DD. The proto-oncogene c-kit and c-kit ligand in human disease (update on cells and cytokines). J Allergy Clin Immunol. 1997;100:435-440.

[24.] Vanderwinden J-M, Rumessen JJ, De Laet M-H, Vanderhaeghen JJ, Schiffmann SN. CD [34.sup.+] cells in human intestine are fibroblasts adjacent to, but distinct from, interstitial cells. Lab Invest. 1999;79:59-65.

[25.] Lasota J, Jasinski M, Sarlomo-Rikala M, Miettinen M. Mutations in exon 11 of c-kit occur preferentially in malignant versus benign GISTs and do not occur in leiomyomas or leiomyosarcomas. Am J Pathol. 1999;154:53-60.

[26.] Ernst SI, Hubbs AE, Przygodzki RM, et al. KIT mutation portends poor prognosis in gastrointestinal stromal/smooth muscle tumors. Lab Invest. 1998;78: 1633-1636.

[27.] Nishita T, Hirota S, Taniguchi M, et al. Familial gastrointestinal stromal tumors with germline mutation of KIT gene. Nat Genet. 1998;19:323-324.

[28.] El-Omar M, Davis J, Gupta S, Ross H, Thompson R. Leiomyosarcoma in leiomyomatosis of the small intestine. Postgrad Med J. 1994;70:661-664.

Accepted for publication May 8, 2000.

From the Departments of Pathology (Drs Wang and French) and Surgery (Dr Vargas), University of California at Los Angeles, Harbor Medical Center, Torrance, Calif.

An abstract reporting in part the results of this study was accepted by the 1999 American Society of Clinical Pathologists/College of American Pathologists Fall National Meeting and published in the American Journal of Clinic Pathology (1999;112;542-543).

Reprints: Samuel W. French, MD, Department of Pathology, 1000 W Carson St, Torrance, CA 90509 (e-mail: french@afp76.humc.edu).
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Author:Wang, Lina; Vargas, Hernan; French, Samuel W.
Publication:Archives of Pathology & Laboratory Medicine
Date:Oct 1, 2000
Words:2725
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