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© 2000 American Society for Clinical Oncology MDR1 Gene Expression and Outcome in Osteosarcoma: A Prospective, Multicenter StudyFrom the Samuel Lunenfeld Research Institute and the University Musculoskeletal Oncology Unit, Mount Sinai Hospital; Departments of Surgery, Public Health Sciences, Medical Genetics and Microbiology, Pathology, and Laboratory Medicine, University of Toronto, Toronto; Department of Orthopaedic Surgery, University of British Columbia, Vancouver, Canada; University of Washington Medical Center, Seattle, WA; Royal Orthopaedic Hospital, Birmingham, United Kingdom; Memorial Sloan-Kettering Cancer Center, New York, NY; and Mayo Clinic, Rochester, MN. Address reprint requests to Jay S. Wunder, MD, 476E-600 University Ave, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; email wunder{at}mshri.on.ca
PURPOSE: Increased expression of the multidrug resistance gene (MDR1) has been implicated in osteosarcoma prognosis. This study represents the first prospective assessment of the prognostic value of MDR1 mRNA expression in patients with newly diagnosed extremity osteosarcoma. PATIENTS AND METHODS: A series of patients with high-grade, nonmetastatic extremity osteosarcoma were enrolled from six tertiary care institutions and observed prospectively for tumor recurrence (median follow-up duration, 30 months). All patients were treated with (neo)adjuvant chemotherapy and surgery. Tumors from 123 patients were analyzed for MDR1 mRNA expression. The association of the level of MDR1 expression with the risk of systemic recurrence was examined using survival analyses with traditional and histologic markers as prognostic factors. RESULTS: Using the highest MDR1 value for each patient, a dose-response relationship was not identified between the level of MDR1 expression and systemic relapse (relative risk, 1.15; P = .44). Analyses based on biopsy or resection values alone gave similar results (P = .11 and .41, respectively, log rank test). In multivariate analysis, large tumor size (> 9 cm) was the only significant independent predictor of systemic outcome (relative risk, 2.8; P = .002). CONCLUSION: We did not identify any correlation between MDR1 mRNA expression and disease progression in patients with osteosarcoma. It is likely that alterations in other genes are involved in resistance to chemotherapy in osteosarcoma and that they play a more critical role than MDR1 in this disease.
THE ADDITION OF chemotherapy to the surgical treatment of osteosarcoma has been the most important recent advance leading to improved patient outcome in this disease.1-3 Although patients with metastases at diagnosis are rarely cured,4 the majority of patients who present with nonmetastatic osteosarcoma can expect a prolonged disease-free survival1-3,5-7. However, 25% to 50% of patients without evidence of metastases at diagnosis subsequently develop systemic disease. Presumably these patients who relapse have drug-resistant tumors. If they could be identified earlier in the course of their disease, they might benefit from alternative treatment strategies. The risk factors for disease progression in osteosarcoma have been studied extensively. Histologic response to preoperative chemotherapy in the resected primary tumor is generally agreed to be the most important predictor of outcome in nonmetastatic osteosarcoma.5-8 A good histologic response with extensive necrosis generally predicts a greater chance of disease-free survival. Other potential predictors of outcome include primary tumor size, primary tumor site, and age at diagnosis.8 Currently, there is no single factor or combination of prognostic factors that can accurately predict tumor responsiveness to chemotherapy or long-term outcome. To address the issue of clinical multidrug resistance in osteosarcoma, a number of studies have examined expression of the multidrug resistance gene (MDR1) in human tumors. P-glycoprotein, the protein product of the MDR1 gene, is thought to function as an adenosine triphosphate–dependent efflux pump that is responsible for the active removal of a wide range of chemotherapeutics, including doxorubicin, from tumor cells.9-11 High levels of expression of the MDR1 gene would therefore be one mechanism whereby drug resistance might develop in a tumor. A proportion of untreated osteosarcomas have been shown to have elevated expression of MDR1 mRNA and P-glycoprotein, which suggests that these tumors may have intrinsic multidrug resistance.12,13 Some, but not all, retrospective studies in osteosarcoma have correlated increased expression of either MDR1 mRNA or protein with a high probability of systemic tumor recurrence.14-17 Overexpression of the MDR1 gene has also been suggested as the cause of chemotherapy failure in several other tumor types.18,19 This report represents the first prospective assessment of the prognostic value of MDR1 mRNA expression in high-grade nonmetastatic osteosarcoma of the extremity. In this study, we evaluated the effect of both MDR1 mRNA expression and standard prognostic indicators on clinical outcome in a cohort of 123 newly diagnosed patients with osteosarcoma.
Patient Eligibility, Treatment, and Clinical Follow-Up One hundred thirty-six patients with high-grade, nonmetastatic osteosarcoma of the extremity were prospectively enrolled from six tertiary care institutions between 1989 and 1994. All patients had newly diagnosed, biopsy-proven osteosarcoma without prior treatment. Patients with pre-existing Paget’s disease, radiation-induced tumors, or previous malignancies (excluding nonmelanoma skin cancer or cervical carcinoma-in-situ) were excluded. Systemic staging studies included total body bone scans and computed tomographic scans of the chest. All patients received pre- and postoperative chemotherapy that included doxorubicin and that was based on one of three protocols that incorporated the following major agents: (1) doxorubicin and cisplatin20; (2) high-dose methotrexate, doxorubicin, and cisplatin6; and (3) ifosfamide, high-dose methotrexate, doxorubicin, and cisplatin.21 The decision regarding which chemotherapy protocol to administer was not based on the presenting patient or tumor characteristics but on the protocol that was in use at each participating institution. Patients with concurrent nonmalignant disease that would preclude the use of one of these standard chemotherapy protocols or those with major chemotherapy protocol violations were also excluded. Definitive surgical treatment of the primary tumor was by wide en bloc excision (n = 114) or amputation (n = 22). The occurrence of local tumor relapse was taken to be an indication of inadequate surgical resection, and these patients were excluded (n = 7). All patients were observed in regular follow-up for a minimum of 24 months from the time of diagnosis or until relapse. No patients were lost to follow-up. Each eligible patient provided a signed consent form before study entry.
Molecular Analyses
Statistical Analysis
In the primary analyses, the highest MDR1 value available from each patient was assessed as a semiquantitative variable, as per the original study protocol. To examine the association of MDR1 levels with the risk of systemic relapse in osteosarcoma, MDR1 was considered alone and then in combination with the other prognostic factors, both with and without an indicator for the doxorubicin/cisplatin chemotherapy protocol. The other prognostic factors included the following: tumor size (
Univariate survival analysis of MDR1 level and each of the prognostic factors was by the log-rank test with Kaplan-Meier survival curves and by the Cox proportional hazards model.24 An ordinal coding scheme was used for MDR1 in which values
Patient and Tumor Characteristics All patients who consented to participate in the study (n = 136) were observed for a minimum of 24 months, and 48 relapses were observed. Seven (53.8%) of 13 specimens that did not have an MDR1 result were from resected tumors that had greater than 90% necrosis after chemotherapy. In comparison, 13 (9.0%) of 145 specimens in this study also had samples from tumors with high necrosis after chemotherapy, but an MDR1 value was obtained for each (P = .001). For these tumors, MDR1 expression ranged from 0.24 to 8.0, with a median value of 2.35. Other patient and tumor characteristics were not significantly different between those with and those without MDR1 values (data not shown). In the remainder of this report, we present only the data for the 123 patients (46 relapses) with MDR1 values. Overall, 65 of these patients (52.8%) were enrolled from centers that used doxorubicin/cisplatin chemotherapy protocols; 29 relapses were observed in this group (Table 1).
MDR1 Expression Levels Based on the highest available MDR1 value for each patient, the level of MDR1 expression was classified as high (ie, it exceeded 2.0) in 44 (35.7%) of the tumor specimens in which a value was obtained and as moderate, between 1.01 and 2.0, in 36 cases (29.3%) (Table 1). Forty-three tumors (35%) had low MDR1 values, with expression  1.0. The MDR1 values ranged from 0.14 to 11.96, with a median value of 1.6 and a 75th percentile of 2.6. MDR1 was not correlated with tumor size, necrosis, site, or chemotherapy protocol (statistical comparisons of proportions were not significant at the conventional 5% level). However, patients with higher MDR1 levels tended to be older (Table 1; P = .04). There was some evidence that the resection samples had higher MDR1 levels than the biopsies. A greater proportion of the resection specimens had MDR1 values in the high category (32 of 79; 40.5%) compared with the biopsy samples (11 of 49; 22.4%; P = .04). In the 22 cases with both biopsy and resection results (Fig 1), the MDR1 level was high in a greater proportion of these resections (11 of 22; 50%) than in their corresponding biopsies (five of 22; 22.7%; P = .03, McNemar’s test), and the distribution of the quantitative MDR1 values was also shifted toward higher values after chemotherapy (P = .02, t test; P = .01, Wilcoxon signed rank test). However, there was no correlation between any of the other prognostic factors and the change in MDR1 expression after chemotherapy.
Prognostic Value of MDR1 and Other Factors When MDR1 was considered alone as an ordinal variable that modelled the risk of relapse as an increasing function of MDR1 level, we did not detect a statistically significant association. The 95% confidence interval indicated that if the true relative risk was greater than 1.65, we would have been able to detect it in this sample (Table 2, univariate models). Although Kaplan-Meier curves that stratified the data by MDR1 level (Fig 2) suggested that patients whose tumors had high MDR1 expression had a worse prognosis than those with intermediate levels, patients with tumors with low MDR1 levels were also associated with a worse prognosis than those with intermediate values, and overall, these differences were not statistically significant (P = .25, log-rank test). Alternative categorization of MDR1 levels based on systematic examination of the data also suggested that patients with extremely low or extremely high values had higher relative risks, but these patients constituted a small proportion of the sample (fewer than 10% in total). Even an optimal ordinal classification (with split points at 2.0 and 4.0), which favorably biases the analysis, did not achieve significance. The negligible relative risk and lack of significance for ordinal MDR1 persisted with nominal MDR1 categorization, as well as with evaluation as a continuous variable. Adjustment for tumor size, necrosis, site, age, and chemotherapy protocol, either singly or together, did not alter the lack of MDR1 significance (Table 2, multivariate model).
The 13 patients for whom an MDR1 value was not obtained had a higher proportion of tumors with greater than 90% necrosis in response to chemotherapy and, therefore, may have been more likely to have had a good prognosis. When we considered these 13 patients to have low MDR1 expression in a univariate survival-sensitivity analysis, the Kaplan-Meier curves were similar to those above and the conclusions were unchanged (P = .2, log-rank test). Because high MDR1 levels were more frequent in resection compared with biopsy specimens, we re-examined the risk of relapse associated with MDR1 level separately for the 49 patients with biopsy specimens and the 79 with resection samples. The Kaplan-Meier curves remained similar to those of the initial analysis, and the conclusions were unchanged (P = .11 and .41 for biopsies and resections, respectively, by log-rank test). Furthermore, for the 22 cases that had both biopsy and resection values (Fig 1), there was no correlation between the change in MDR1 category (ie, low, moderate, or high) or the change in quantitative level of expression and clinical outcome.
In univariate analyses of the other prognostic factors, there was evidence that tumor size and necrosis (
Importance of MDR1 in Chemotherapy Subgroups Using interaction terms in the Cox proportional hazards model and likelihood ratio tests, we did not detect evidence for variation in the importance of MDR1 across chemotherapy protocols (data not shown). In addition, we repeated all univariate and multivariate analyses for the 65 patients who received the doxorubicin/cisplatin protocol. Similar results were obtained for MDR1 (Fig 5), and there was no evidence of a linear relationship between MDR1 levels and the risk of systemic relapse in this subgroup (relative risk, 0.96; P = .88; 95% confidence interval, 0.58 to 1.60).
Unexpectedly, we found no relationship between the level of MDR1 expression and systemic relapse in patients with osteosarcoma. Our results, therefore, do not support the hypothesis that progressively increasing levels of MDR1 mRNA should correlate with the multidrug resistance phenotype.9-11 In this study, we found that osteosarcomas had various levels of MDR1 mRNA expression, with 35.7% of tumors having high levels. This is similar to the proportion of tumors with elevated P-glycoprotein levels identified by immunohistochemistry in other investigations.15-17,26 However, our findings do not support the results of studies that showed retrospectively that elevated P-glycoprotein levels correlated with a poor prognosis in osteosarcoma.15,16 Our results are more consistent with investigations that failed to identify a significant correlation between patient outcome or response to chemotherapy and P-glycoprotein status in osteosarcoma.17,26,27 One possible reason for these conflicting results is that the methods used for detection of MDR1 mRNA or P-glycoprotein differ in sensitivity, and various thresholds have been defined as positive. However, most methodologic studies that compared different detection techniques have found a correlation between levels of MDR1 mRNA and P-glycoprotein and the degree of drug resistance in a variety of cell lines and tumors.28-34 The RT-PCR assay used in this study has been shown to be sensitive for quantitative analysis of low levels of MDR1 mRNA expression and can reliably detect one copy of MDR1 mRNA per cell.12,13 For ovarian and small-cell lung cancers, this low level of MDR1 mRNA has been found to correlate with clinical resistance to chemotherapy.19 In contrast, a higher sensitivity threshold has been demonstrated for detection of P-glycoprotein by immunohistochemistry.28-35 Our group previously reported that immunohistochemical detection of P-glycoprotein in osteosarcoma was less sensitive than measurement of MDR1 mRNA by RT-PCR.35 Based on our experience with these two techniques, few tumors in this study would have been predicted to have sufficiently elevated MDR1 mRNA to allow P-glycoprotein detection by immunohistochemistry. Immunohistochemical studies have suggested that a relatively small number of tumor cells within a primary osteosarcoma overexpress P-glycoprotein and that these few cells are responsible for the MDR phenotype.15-17,26,27 Cellular heterogeneity of MDR1 expression could be overlooked using the RT-PCR assay. That is, the mRNA from a few highly expressing cells within a tumor sample could be diluted with RNA from lower or nonexpressing cells, and the tumor would be considered to have a lower MDR1 mRNA level. If there is heterogeneity of MDR1 mRNA/P-glycoprotein expression in primary osteosarcomas and high levels are associated with systemic disease, then (1) MDR1 levels in postchemotherapy resection specimens, which are more frequently in the high category than levels in biopsies, might be expected to correlate best with clinical outcome and (2) metastases would be expected to express higher MDR1 levels than the tumors from which they arose. Contrary to the above expectations, we found that neither resection nor biopsy levels of MDR1 correlated with the development of systemic disease in our study. Furthermore, we previously showed that in the majority of cases, primary and metastatic tumors from the same patient exhibited similar levels of MDR1 mRNA.13 The distribution of MDR1 mRNA levels in metastatic osteosarcoma specimens was similar to that expressed in primary tumors, which indicates that not all metastases have high levels of MDR1 expression. There are a number of possible explanations for the finding that MDR1 expression was higher in tumor resections compared with biopsies. Preoperative chemotherapy may have caused a further induction of intrinsic MDR1 gene expression or newly acquired expression in some cases. Alternatively, chemotherapy may have killed a proportion of the drug-sensitive cells, leaving behind the resistant cells that presumably would have higher MDR1 levels. Regardless of the explanation, neither the biopsy nor resection MDR1 values correlated with clinical outcome. Although paired biopsy and resection specimens were only examined for 22 patients (Fig 1), the lack of correlation between the change in MDR1 expression after chemotherapy and systemic relapse for these patients further supports the results of this study. In other tumors, immunohistochemical analysis of P-glycoprotein expression relative to patient outcome has also produced contradictory results. In a study of 30 children with rhabdomyosarcoma and undifferentiated soft tissue sarcoma, Chan et al18 showed that P-glycoprotein expression was strongly correlated with both response to chemotherapy and prognosis. However, in a larger study of 76 patients with rhabdomyosarcoma, P-glycoprotein expression did not predict for poor clinical outcome.36 Interestingly, the latter study used three different monoclonal antibodies against P-glycoprotein and found the following: (1) each antibody identified a different subset of P-glycoprotein positive tumors, (2) results were different for each antibody depending on the immunohistochemical detection system, and (3) depending on the criteria used to define positivity, in some instances detection of P-glycoprotein was even a favorable factor for survival.36 These conflicting reports illustrate some of the difficulties associated with immunohistochemical analysis of P-glycoprotein, especially in solid tumors, as recently highlighted by the P-glycoprotein Methods Detection Workshop.30 Furthermore, a number of the commonly used anti–P-glycoprotein antibodies, (eg, C219,37,38 C494,39 and JSB-140) cross-react with other cellular proteins, limiting their specificity and, therefore, usefulness. Patients in this study were treated with one of three different chemotherapy protocols, depending on which was being used at their treating institution.5,20,21 Each protocol included doxorubicin, which is a substrate for P-glycoprotein–mediated transport and one of the most effective agents against osteosarcoma. The inclusion of additional drugs in these regimens that are not substrates for P-glycoprotein, including methotrexate, cisplatin, and ifosfamide, might help explain the surprising results of this study. That is, these additional agents could possibly bypass the drug-resistant effect of elevated MDR1 levels. If this were the case, the two-drug protocol of doxorubicin and cisplatin should have been the most likely to show a positive MDR1 effect if it did exist. However, the overall results remained unchanged when the 65 patients who received only this protocol were analyzed. Examination of traditional prognostic factors in multivariate analysis revealed that only tumor size correlated significantly with patient outcome (relative risk, 2.80; P = .002). In this study, tumor size was measured in one dimension as an estimate of local tumor burden.41 It is likely that tumor volume itself may have been of even more predictive value.42 This would support the Goldie-Coldman hypothesis that larger tumors are at higher risk of failing to respond to chemotherapy because of the presence or development of drug resistance.43 Chemotherapy-induced necrosis is likely another indirect measure of tumor chemosensitivity and has been considered the best available predictor of systemic relapse in osteosarcoma.5-7,20 In this study, it was of borderline significance in univariate analysis. Although the relative risk estimate for necrosis was greater than 2, the confidence interval was wide, which suggests low power in this sample. This result was somewhat surprising because few studies in osteosarcoma have failed to identify a significant correlation between necrosis and outcome.44 This finding is likely related to the interobserver variability of different pathologists at each participating institution in defining necrosis in this multicenter study. In comparison, central pathology review was part of other multicenter studies that did identify a significant correlation between necrosis and outcome.7,20 We did not identify a correlation between MDR1 level and chemotherapy-induced tumor necrosis. Interestingly, neither of the two studies that identified a positive relationship between P-glycoprotein expression and patient outcome in osteosarcoma found any correlation between these two variables.15,16 This suggests that even if P-glycoprotein does play a role in chemotherapy resistance, it may also be an indicator of another unrelated phenotype such as local tumor aggressiveness, as was recently suggested for colon carcinoma.45,46 If MDR1 expression is important in osteosarcoma, it is likely that there are also other mechanisms that may play a more critical role in this disease. Mutation of the p53 gene might be another potential mechanism that affects outcome.47 Mutant p53 has been suggested to cause tumor drug resistance and more aggressive tumors.48-51 In addition, altered p53 proteins may stimulate the MDR1 promoter, whereas wild-type p53 may act as a repressor.52-54 The interaction between these two genes may be important and is also being investigated.
Supported by grant no. 010450 from the National Cancer Institute of Canada, Toronto, Canada (J.S.W., R.S.B., and I.L.A.). We thank Carmen Mak and Joseph Gao for expert statistical analysis.
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ABSTRACT
INTRODUCTION
2 test for independent samples. In 22 cases with both biopsy and resection results, the MDR1 categories were compared using McNemar’s test for paired samples, and the quantitative values were compared with a paired t test and a Wilcoxon signed rank test. 
