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© 2000 American Society for Clinical Oncology Soft Tissue Leiomyosarcomas and Malignant Gastrointestinal Stromal Tumors: Differences in Clinical Outcome and Expression of Multidrug Resistance ProteinsFrom the Departments of Pathology, Surgical Oncology, and Medical Oncology, University Hospital Groningen, and Department of Medical Genetics, University of Groningen, Groningen; and Department of Pathology, Free University Hospital, Amsterdam, the Netherlands. Address reprint requests to Boudewijn E.C. Plaat, MD, PhD, Department of Pathology, University Hospital Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands; email b.e.ch.plaat{at}kno.azg.nl
PURPOSE: Several studies have reported clinical behavior and chemotherapy resistance in leiomyosarcomas, but these studies did not differentiate between soft tissue leiomyosarcomas (LMS) and malignant gastrointestinal stromal tumors (GIST). Multidrug resistance (MDR) has been associated with the expression of P-glycoprotein (P-gp), multidrug resistance protein (MRP1), and lung resistance protein (LRP). The aim of the present study was to compare LMS and GIST with respect to clinical outcome and MDR parameters. PATIENTS AND METHODS: Clinical outcome was evaluated in 29 patients with a primary deep-seated LMS and 26 patients with a primary malignant GIST. Paraffin-embedded material, available for 26 patients with LMS and 25 with GIST, was used for immunohistochemical detection of P-gp, MRP1, LRP, and c-kit. RESULTS: Mean overall survival (OS) was 72 months for LMS patients and 31 months for GIST patients (P < .05). Metastases occurred in 16 (59%) of 27 assessable LMS patients and in 10 (56%) of 18 assessable GIST patients. LMS predominantly metastasized to the lungs (14 of 16 patients), whereas GIST tended to spread to the liver (five of 10 patients) and the abdominal cavity (three of 10 patients; P < .001). P-gp and MRP1 expression was more pronounced in GIST than in LMS (P < .05): the mean percentage of P-gp expressing cells was 13.4% in patients with LMS and 38.4% in patients with GIST, and the mean percentage MRP1 expressing cells was 13.3% in patients with LMS and 35.4% in patients with GIST. LRP expression did not differ between LMS and GIST. c-kit was expressed in 5% of the LMS patients and in 68% of the GIST patients. CONCLUSION: LMS patients have a better survival than GIST patients, and the metastatic pattern is different. Expression of MDR proteins in LMS is less pronounced than in GIST.
LEIOMYOSARCOMAS (LMS) are a group of malignant tumors that arise from tissue of mesenchymal origin and have histologic features consistent with smooth-muscle differentiation. They account for approximately 10% of all soft tissue sarcomas (STS).1,2 However, it has been suggested that the tumors with smooth-muscle differentiation that arise in the digestive tract are a distinct tumor entity derived from the interstitial cells of Cajal, although the histogenesis of these malignant gastrointestinal stromal tumors (GIST) is still the subject of debate.3-12 Expression of the c-kit proto-oncogene is found in a large number of GIST and might play a critical role in their oncogenesis.13,14 In several studies, metastatic LMS were reported to respond poorly to cytostatic drugs, but in most of the clinical studies, a distinction between LMS and GIST was not made.15-18 In the group of STS, the most common site of first metastasis is the lungs. Lung metastases respond better to chemotherapy than metastases at other sites.19 Whether LMS and GIST differ in the development of liver metastases, which are associated with a poor prognosis, at present is unknown. Multidrug resistance (MDR), ie, resistance to numerous structurally unrelated natural product drugs such as the widely applied anthracyclines, has been associated with the overexpression of proteins like the MDR-1 gene product P-glycoprotein (P-gp)20-28 and the multidrug resistance protein-1 (MRP1) in drug-resistant cell lines and neoplastic tissue.27,29-33 Several studies indicate a possible role in clinical drug resistance for another protein: the major vault protein or lung resistance protein (LRP).30,34-39 Expression of the MDR-associated proteins P-gp, MRP1, and LRP has not been systematically studied in LMS and GIST, although this might reveal clinically valuable information. The present study of a group of 29 patients with LMS and 26 patients with GIST was performed to determine whether LMS and GIST are different clinical entities with regard to clinical behavior and MDR protein expression.
Clinical data (ie, patient characteristics, location of the tumor, presence of metastatic disease, follow-up status, and survival data) were obtained by the analysis from the hospital medical records (University Hospital Groningen) or information of the general practitioners. Between 1980 and 1998, 55 consecutive cases were evaluated in the University Hospital Groningen: 29 patients with a deep soft tissue LMS and 26 patients with an LMS of the digestive tract or GIST (Table 1). Sixteen LMS (55%) were located in the lower extremity, six (21%) in the upper extremity, four (14%) in the retroperitoneum, and three (10%) in other parts of the body. Twelve malignant GISTs (46%) were derived from the stomach, six (23%) from the small intestine, six (23%) from the large intestine, one (4%) from the esophagus, and in one patient (4%), the tumor presented as a large intra-abdominal mass suspected to derive from the stomach. Paraffin-embedded tumor material was available from 26 patients with LMS of soft tissue and 25 patients with malignant GIST. Patients with superficial LMS (ie, originating from the cutis or subcutis) were not included, because it is reported that such patients have a better prognosis than patients with deep soft tissue LMS.1 For this study, only patients with previously untreated, primary tumors were included. In all cases, the histologic diagnoses were confirmed by central revision of hematoxylin-eosin–stained paraffin sections with or without additional immunohistologic stains.1,5 The 29 LMS were graded according to the grading system of Coindre et al,40 which resulted in eight grade 1 (28%), 14 grade 2 (48%), and seven grade 3 LMS (24%). A GIST was considered malignant if there was both mitotic activity (ie,  one mitotic figure per 2 mm2) as well as a tumor size of more than 5 cm in diameter, according to the recommendations of Suster.5
MDR Protein and c-kit Expression For detection of P-gp, the monoclonal antibody C494 (Signet Laboratories, Dedham, MA) was used in a concentration of 120 µg/mL in phosphate-buffered saline (PBS; Merck, Darmstadt, Germany) plus 1% bovine serum albumin (BSA; Serva Electrophoresis GmbH, Hamburg, Germany). For detection of MRP1, the monoclonal antibody MRPr1 (provided by R.J.S., Department of Pathology, Free University Hospital, Amsterdam) in a concentration of 20 µg/mL in 1% BSA/PBS was used. For detection of LRP, the monoclonal antibody LRP (Transduction Laboratories, Los Angeles, CA) in a concentration of 250 µg/mL in 1% BSA/PBS was used. As controls for immunohistochemical detection, cytospins of the well-documented cell lines A2780 and GLC4 and their corresponding multidrug resistant sublines (ie, overexpressing P-gp [A2780 AD], MRP1 [GLC4/ADR], and LRP [GLC4/ADR])41,42 as well as paraffin-embedded formalin-fixed liver (P-gp), lung (MRP1), and colon (LRP) tissue were used. Paraffin sections (3 µm) were placed on positively charged glass slides and were dried. Immunohistochemistry was performed according to a method modified from Shi et al43 and Emanuels et al.44 Briefly, after heating on a hot plate, the slides were dewaxed in xylene and rehydrated in serial ethanol washes (100%, 96%, and 70%). After each of the subsequent steps, three 5-minutes washes in PBS were carried out. After heating three times in an autoclave for 5 minutes at 115°C in a 20 mmol/L blocking reagent (Boehringer Mannheim, Mannheim, Germany) with pH = 6.0, endogenous peroxidase activity was blocked by incubation with 1% hydrogen peroxide in PBS for 30 minutes. The slides were incubated with the specific antibody in 1% BSA/PBS (pH = 7.4) for 1 hour at room temperature in a humidified chamber. The primary antibody was detected with a rabbit antimouse (DAKO, Glostrup, Denmark; C494 and LRP) or rabbit antirat (DAKO; MRPr1) peroxidase-labeled secondary antibody diluted 1:50 in BSA/PBS + 1% human serum (type AB) followed by incubation with goat antirabbit conjugated peroxidase diluted 1:50 in BSA/PBS +1% human serum (type AB). 3.3-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) with imidazole (Merck) in PBS was used as the chromogen according to the manufacturer’s instructions. After counterstaining with Mayer’s hematoxylin, the slides were dehydrated through graded alcohols and mounted with coverslips. For detection of c-kit, the polyclonal antibody c-kit (Santa Cruz Biotechnology, Santa Cruz, CA) in a concentration of 200 µg/mL in 1% BSA/PBS was used. As controls for immunohistochemical detection, paraffin-embedded formalin-fixed colon tissue was used. Paraffin sections (3 µm) were placed on positively charged glass slides and were dried. After heating on a hot plate, the slides were dewaxed in xylene and rehydrated in serial ethanol washes (100%, 96%, and 70%). After each of the subsequent steps, three 5-minutes washes in PBS were carried out. After one night of incubation in 0.1 mol/L tris(hydroxymethyl)aminomethane hydrochloride (Merck) at 80°C with pH = 6.0, endogenous peroxidase activity was blocked by incubation with 1% hydrogen peroxide in PBS for 30 minutes at room temperature. After blocking endogenous biotin and avidin for 15 minutes with Avidin/Biotin Blocking Kit (Vector Laboratories Inc, Burlingame, CA) the slides were incubated with the polyclonal c-kit antibody (Santa Cruz Biotechnology) in 1% BSA/PBS for 1 hour at room temperature in a humidified chamber. The primary antibody was detected with a sheep antirabbit (DAKO) biotinylated secondary antibody diluted 1:300 in 1% BSA/PBS + 1% human serum (type AB) for 30 minutes followed by incubation streptavidin biotinylated horseradish peroxidase (StreptAB-complex-HRP; DAKO) diluted 1:100 in BSA/PBS +1% human serum (type AB) for 30 minutes. 3.3-diaminobenzidine tetrahydrochloride (Sigma) with imidazole (Merck) in PBS was used as the chromogen according to the manufacturer’s instructions. After counterstaining with Mayer’s hematoxylin, the slides were dehydrated through graded alcohols and mounted with coverslips.
The expression of P-gp, MRP1, and LRP was assessed independently by two observers (B.P, H.H.) without knowledge of the definitive histologic diagnosis or clinical data. The P-gp, MRP1, and LRP proteins were studied in adjacent slides of the most viable parts of the tumor. Amounts of P-gp, MRP1, and LRP expression were semiquantitatively assessed by estimating the percentage of positive stained tumor cells. According to the literature, tumor samples were considered negative for expression of each of the proteins if
Statistics
Patient Characteristics and Clinical Outcome As listed in Table 1, the LMS group consisted of 12 men and 17 women, with a mean age of 57 years (SD, 16.2; range, 20 to 83 years). The GIST group consisted of 13 men and 13 women, with a mean age of 58 years (SD, 14.7; range, 18 to 80 years). Sex and age were equally distributed between both groups. OS of the 29 patients with LMS (mean, 72 months; median, 40 months) was significantly (P < .05) better than the OS of the 26 patients with a GIST (mean, 31 months; median, 28 months; Fig 1). The 2-year and 5-year OS rates in the group of LMS patients were 73.9% and 41.2%, respectively, whereas the 2-year and 5-year OS rates were significantly lower in the group of GIST patients (58.5% and 13.0%, respectively).
In the GIST group, OS was not significantly influenced by the location of the primary tumor; ie, patients with malignant GIST located in the stomach had a mean OS of 29 months (median, 28 months), whereas mean OS times for small-intestine and large-intestine GIST were 24 (median, 28) and 41 (median, 20) months. Because of the long period in which the patients were studied, it was not possible to determine whether there had been distant metastases in two LMS cases and eight GIST cases. Of the remaining 27 LMS patients, 16 developed metastases: lung metastases were found in 14 patients and skin metastases in two patients (Table 2). None of the LMS patients developed liver metastases as the first metastatic site, but four patients developed liver metastases subsequent to other distant metastases. Of the remaining 18 GIST patients, 10 patients developed distant metastases: liver metastases were found in five patients, intra-abdominal metastases in three patients, and regional lymph node metastases in one patient. One GIST patient developed intrathoracic metastases, presumably in conjunction with intra-abdominal metastasis. Although the frequency of metastases in the assessable LMS (16 of 27) and GIST (10 of 18) patients was comparable, the liver was the most frequent first metastatic site in GIST patients (P < .01), whereas the lungs were more commonly involved in patients with deep-seated LMS (P < .001).
P-gp, MRP1, and LRP expression in LMS and GIST P-gp and MRP1 were significantly more expressed in the GIST than in the LMS: P-gp–positive LMS were found in eight of 26 cases (31%) and P-gp–positive GIST could be identified in 17 of 25 cases (68%; P < .05). MRP1-positive LMS were found in eight of 26 cases (31%) and MRP1-positive GIST could be identified in 17 of 25 cases (68%; P < .05; Table 3). As shown in Fig 2, the amount of P-gp– and MRP1-expressing tumor cells was significantly higher in GIST than in LMS (both P < .05; Table 4). LRP expression did not differ between LMS and GIST but was more often expressed than P-gp and MRP1 (both P < .001). A minority of LMS (12%) and GIST (12%) had no MDR expression, whereas 12% of the LMS and 48% of the GIST expressed all three MDR proteins (P < .01). Numbers of tumors with coexpression of P-gp, MRP1, or LRP are listed in Table 3.
A correlation (r = 0.33; P < .05) was found between P-gp and MRP1 expression in the whole group of tumors, but not in the subgroups of LMS and GIST. LRP expression was not related to P-gp or MRP1 expression. P-gp, MRP1, and LRP expression were not correlated with tumor grade nor with mitotic activity.
OS in Relation to MDR Status in LMS and GIST
MDR Status and OS in Relation to c-kit Expression Of 21 of 29 LMS patients who were assessable for c-kit expression, one (5%) showed c-kit expression, whereas of 22 of 26 GIST patients who were assessable, 15 (68%) showed c-kit expression (P < .001; Table 6). There was no difference in OS between c-kit–positive or c-kit–negative tumors (all cases). Furthermore, no differences in OS were observed between c-kit–positive GIST (mean, 30 months; median, 28 months) and c-kit–negative GIST (mean, 19 months; median, 20 months). P-gp and MRP1 were more expressed (P < .001) in the 16 c-kit–positive tumors than in the 27 c-kit–negative tumors. LRP expression did not differ between c-kit–positive or c-kit–negative tumors. Number of mitotic figures was not correlated with c-kit status.
Immunohistochemical and molecular (cyto)genetic findings provide evidence that GIST should be considered as a specific group of neoplasms.5,46 GISTs have been diagnosed as smooth-muscle tumors of the digestive tract for many years, and the studies evaluating clinical outcome and treatment response have classified them as LMSs of the digestive tract. A number of studies have reported the chemoresistance of the so-called LMSs and the poor prognosis of patients with an abdominal LMS, but it is not clear whether these nonresponding malignant smooth-muscle neoplasms are GIST, LMS, or uterine LMS.15-18 The results of a recent study by Edmonson et al,47 in which malignant GIST and other LMS were treated with dacarbazine, mitomycin, doxorubicin, and cisplatin, indicate that GISTs are more chemotherapy-resistant than other neoplasms with smooth-muscle differentiation. Although complete follow-up data could not be obtained in two of 29 LMS cases and in eight of 26 GIST cases, even in this selected number of patients, the differences in metastatic pattern were significant. The present study showed that equal percentages of LMS and GIST patients develop distant metastases. However, the frequency of lung metastases in metastasized LMS was 88% and the frequency of liver metastases was 25%, whereas lung metastases developed in 10% of the metastasized GIST patients and liver metastases were found in 50%. This suggests the idea that studies reporting the high incidence of hepatic metastases in LMSs18,48 might evaluate malignant GIST and that (metastatic) LMS might be more drug-sensitive than has been suggested in earlier studies. No previous studies were performed in which GISTs were compared with LMSs of deep soft tissue regarding clinical outcome and the presence of drug-resistance–associated proteins. The results of the current study not only indicate that patients with an LMS have a better prognosis than patients with malignant GIST but revealed clinically important evidence that malignant GIST express P-gp and MRP1 more frequently than LMS.
The OS was evaluated without information of the adequacy of the primary surgical treatment and of additional treatment, because in the accrual of a relatively large amount of patient and tumor data of relatively rare neoplasms, a 19-year period is involved, making the importance of the additional information of the various applied treatment regimens questionable. In the present study, LMSs have a better clinical behavior than malignant GISTs. Several studies reported the 5-year survival rate in GIST to be in the range of 20% to 78%8 but did not clearly indicate whether these GISTs were malignant or benign. In our study, we combined the presence of both mitotic activity (ie, The finding that GISTs have significantly more expression of P-gp and MRP1 than LMSs indicates that GIST is not only a different tumor, but is also of therapeutic importance. In most STSs, doxorubicin-based regimens are administered in metastatic disease, but also in the adjuvant setting.51-59 Edmonson et al47 showed that GISTs are more chemotherapy-resistant than other malignant smooth-muscle tumors when doxorubicin-based chemotherapy is used. However, doxorubicin resistance is related to P-gp and MRP1,30 and the results of our study strongly imply that especially in malignant GIST with high expression of both P-gp and MRP1, doxorubicin is not indicated, and new non-MDR related cytostatic agents are awaited. Both P-gp and MRP1 expression are frequently detected in normal tissue of the digestive tract,60,61 which could explain their expression in GIST. Because P-gp expression and MRP1 expression were correlated neither to tumor grade in LMS nor to the mitotic activity in both LMS and GIST, it seems that neither MDR parameter is related to tumor aggressiveness and that MDR parameters are more related to the histologic type and the tissue from which they derive, as has been demonstrated in other studies.61-67 High LRP expression was found both in LMSs and malignant GISTs, and LRP expression did not have prognostic value in either group. The high expression of LRP might be due to the fact that LRP is present in the epithelial tissues of the digestive tract. Although the distribution of LRP expression in normal tissues has not been studied extensively, it was found to be present in muscle tissue, but not in muscle tissue with high MRP1 expression or in normal smooth muscle.37,68 In the present study, no inverse correlation between MRP1 and LRP expression was found, which indicates that LRP expression has not been decreased by simultaneous MRP1 expression in these smooth-muscle neoplasms. On microscopic examination, the LRP staining was mostly cytoplasmic and normal smooth muscle showed no clear staining. The exact function of LRP is not yet understood, although there is evidence that LRP acts as an intracytoplasmic transporter of cytotoxic agents. In contrast to acute myeloid leukemia and ovarian cancer where LRP expression has been found to be an independent prognostic factor for response to chemotherapy and outcome,34-36 not much is known about LRP expression in STS, and its prognostic role in STS is still unclear. Other studies have shown the clinical importance of MDR-protein (co)expression in sarcomas in general.25,27,31,69-73 However, the analysis of MDR protein expression can be hampered in various ways. For instance, differences in tissue preparation and storage as well as the use of different techniques and monoclonal antibodies might lead to diverse results.74 Although immunohistology can visualize which parts of the tumor and which tumor cells express the different MDR proteins, Oda et al28 found that only 47% of the MDR-1 mRNA–positive tumors expressed P-gp as determined by immunohistologic detection. Furthermore, it seems that not all immunohistologically detected P-gp expressing tumors have functional P-gp.75 This discrepancy between the amount of reverse transcriptase polymerase chain reaction–detected MDR-1 mRNA, the immunohistologically detected P-gp expression, and the functional P-gp efflux pump illustrates the problem of interpreting MDR protein expression. The antibodies used in the present study were selected after adequate analyses of well-characterized cell lines, Western blot analysis, and the influence of various formalin fixation periods on the immunohistologic detection as well as the immunohistologic detection in formalin-fixed paraffin-embedded control tissues. The cutoff level of 5% was used to assess whether tumors were P-gp–, MRP1-, or LRP-positive or -negative. A cutoff level was introduced earlier34 because immunohistologic assessment cannot determine accurately if less than 5% stained cells in a minority of tumor specimens are indeed malignant tumor cells or preexistent normal tissue, blood cells, or endothelial cells. The expression of c-kit in GIST and LMS is comparable to that found in other studies.10,14,76-78 The expression of c-kit was not associated with OS or mitotic activity, indicating that c-kit is more associated with oncogenesis than with tumor aggressiveness. The observed relation of c-kit expression with both P-gp and MRP1 expression has not been observed before in solid tumors, but the association between c-kit and P-gp has been reported in acute myeloid leukemia.79 Because some authors only use the diagnosis of GIST for c-kit overexpressors, the finding of high P-gp and MRP1 expression in c-kit–positive tumors strengthens the idea that GISTs indeed have a higher level of drug resistance than LMSs. Further molecular studies should establish the biologic significance of the observed relation between the c-kit proto-oncogene and membrane drug transporters P-gp and MRP1 in these aggressive gastrointestinal mesenchymal tumors. In conclusion, LMS patients have a better survival than GIST patients, and LMSs have a less pronounced drug resistance pattern than GISTs. Therefore, studies evaluating chemotherapeutic regimens should differentiate between LMS and malignant GIST. The observed high expression of P-gp and MRP1 in GISTs might explain the poor response of such tumors to standard (anthracycline-based) chemo- therapy. Although an equal number of LMSs and GISTs develop distant metastases, LMSs predominantly metastasize to the lungs, whereas GISTs tend to spread to the liver and the abdominal cavity. Further studies are warranted to determine whether new cytostatic agents are effective in GIST and might be used in case of metastatic disease.
Supported by the Dutch Cancer Society (Koningin Wilhelmina Fonds) grant no. RUG 95-1085.
Presented in part at the Thirty-Fifth Annual Meeting of the American Society of Clinical Oncology, Atlanta, GA, May 13-18, 1999.
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ABSTRACT
INTRODUCTION
5% of the cells were positive.
