Background Increased levels of P-glycoprotein occur in someosteosarcomas. In this study we determined the relation betweenP-glycoprotein status and outcome in patients with high-gradeosteosarcoma.
Methods P-glycoprotein status was determined immunohistochemicallyin specimens of osteosarcoma of the extremities (stage II) from92 patients who were treated with surgery and chemotherapy.The P-glycoprotein status was analyzed in relation to the lengthof event-free survival.
Results The presence of increased levels of P-glycoprotein inthe osteosarcoma was significantly associated with a decreasedprobability of remaining event-free after diagnosis (P = 0.002).In a multivariate analysis, P-glycoprotein status (P = 0.001)and the extent of tumor necrosis after preoperative chemotherapy(P = 0.04) were independent predictors of clinical outcome.The risk of adverse events was increased substantially (rateratio, 3.37; 95 percent confidence interval, 1.60 to 7.10) amongpatients with increased levels of P-glycoprotein in tumor cells,as compared with patients who did not have increased levelsof P-glycoprotein in tumor cells.
Conclusions In patients with high-grade osteosarcoma treatedwith surgery and chemotherapy, the presence of increased levelsof P-glycoprotein in tumor cells is associated with a significantlyincreased risk of adverse events and is independent of the extentof necrosis after preoperative chemotherapy.
The resistance of tumors to multiple drugs is a major problemin cancer chemotherapy. P-glycoprotein, a transmembrane ATP-dependentefflux pump encoded by the mdr1 gene,1 has a central role inmultidrug resistance in vitro,2 and its clinical relevance isunder investigation.3,4 Increased amounts of P-glycoproteinmay confer multidrug resistance on cells by preventing the intracellularaccumulation of a variety of cytotoxic drugs,5 including doxorubicin,the most effective agent for the treatment of osteosarcomas.
Drug resistance may represent an important prognostic factorin osteosarcoma, the most frequent primary malignant bone tumor.Although the outcome of osteosarcoma has improved considerablysince the addition of chemotherapy to regimens involving surgery,systemic relapses still occur in 40 to 50 percent of cases.6Preliminary observations have shown a wide range of P-glycoproteinlevels in osteosarcomas,7,8,9 and a trend toward a poorer outcomein patients whose tumors express high levels of the mdr1 gene.8,9Moreover, as compared with the primary lesions, a significantlyhigher proportion of the metastases of osteosarcoma were resistantto multiple drugs.9 The ability to predict the course of osteosarcomaby identifying patients who will not respond to conventionalchemotherapy would help guide treatment, allowing the inclusionof agents capable of modulating multidrug resistance.10,11,12,13,14
We sought to determine whether the presence of increased levelsof P-glycoprotein is a predictor of the clinical behavior ofosteosarcoma. We studied 92 patients with high-grade osteosarcomaof the extremities who were treated with surgery and chemotherapy.
Methods
Patients and Tumors
Between September 1986 and December 1989, 319 patients withnewly diagnosed osteosarcomas were seen at the Rizzoli Institutein Bologna, Italy. Of these, 67 did not have conventional high-gradeosteosarcoma (26 had malignant fibrous histiocytoma of bone;11, parosteal osteosarcoma; 7, periosteal osteosarcoma; 7, low-gradecentral osteosarcoma; 6, post-radiation osteosarcoma; 6, osteosarcomaas part of dedifferentiated chondrosarcoma; and 4, osteosarcomaarising in Paget's disease), 33 had evidence of metastases,24 did not have osteosarcoma of the extremities, 7 were olderthan 40 years, and 1 had received treatment elsewhere. Of theremaining 187 patients who were considered eligible for a clinicalstudy whose aim was to optimize chemotherapy for osteosarcoma,1523 declined to participate and were treated with surgery followedby chemotherapy. The clinical and pathological characteristicsof the remaining 164 patients have been described previously.15
The present study included 92 patients for whom tumor samplesfrom the biopsy specimens (obtained before chemotherapy andpreserved in archival paraffin-embedded tissue blocks) wereavailable for immunohistochemical analysis. Biopsy specimensfrom the remaining 72 patients were inadequate for P-glycoproteinevaluation (47 patients underwent a needle biopsy and 25 patientshad been given a diagnosis elsewhere). Table 1 summarizes theclinical and pathological characteristics of the 92 patientswith adequate tissue for analysis and compares them with theentire group of 164 patients. There were no significant differencesbetween the two groups, indicating that the subgroup of 92 patientswhose tumors were analyzed for P-glycoprotein was representativeof the group as a whole. All tumors were classified as stageII conventional high-grade osteosarcomas.16,17 The tumors wereclassified as intracompartmental (stage IIA) in 2 patients andextracompartmental (stage IIB) in 90 patients.
Table 1. Clinical and Pathological Features of the Subgroup of 92 Patients with Osteosarcoma in Whom P-Glycoprotein Immunoreactivity Was Assessed and of the entire Group of 164 Patients with Osteosarcoma.
Chemotherapy was given before and after surgery.15 The preoperativeregimen included two cycles of methotrexate (8 g per squaremeter of body-surface area) plus leucovorin calcium (180 mg),cisplatin (120 mg per square meter), and doxorubicin (60 mgper square meter). In each case, the surgical procedures tookinto account the location and extent of the tumor and the lifeexpectancy of the patient. A limb-salvage procedure was performedin 83 patients (90 percent), whereas 9 (10 percent) underwentamputation. The surgical margins of the tumor specimens werehistologically defined according to the system of Enneking etal.17 The margins were radical in 8 patients (9 percent), widein 73 (79 percent), marginal in 5 (5 percent), and intralesionalin 6 (7 percent). The extent of tumor necrosis was evaluatedaccording to a previously described semiquantitative method.18The response to chemotherapy was considered good if the extentof tumor necrosis was 90 percent or greater and poor if it wasless than 90 percent. Sixty-seven patients had a good response(73 percent), whereas 25 had a poor response (27 percent). Postoperativechemotherapy for those with a good response included a 21-weekcourse of the same drugs used preoperatively, whereas patientswith a poor response received a 30-week regimen that also containedifosfamide (2 g per square meter) and etoposide (120 mg persquare meter). During and after postoperative chemotherapy,the patients underwent clinical and radiographic evaluationof the operated-on limb and the chest every two months. Aftercompleting chemotherapy, the patients were seen every two monthsfor the first two years, every four months during the thirdyear, and every six months thereafter.
Adverse events were defined as a recurrence of the tumor atany site, local or systemic, or death during remission. Event-freesurvival was calculated from the date of the initial diagnosis.All patients were followed for a minimum of 63 months (median,72). Results were updated in December 1994: 64 patients (70percent) were free of disease, whereas 28 (30 percent) had pulmonarymetastases.
Monoclonal Antibodies and Immunohistochemical Analysis
Monoclonal antibodies C219 (Centocor, Malvern, pa.), MRK16 (KamiyaBiomedical, Thousand Oaks, Calif.), and JSB-1 (Sanbio, Uden,the Netherlands) recognize different, mutually exclusive epitopesof P-glycoprotein.19,20,21 Optimal conditions for P-glycoproteinimmunostaining were established on sections obtained from thedrug-sensitive U-2 OS human osteosarcoma cell line and its multidrug-resistantderivative,22 U-2 OS/DX30. The drug resistance of U-2 OS/DX30is 15 times higher than that of its parent; this is a low levelof resistance that is representative of that generally presentin clinical settings.23 For P-glycoprotein immunostaining, thecells were pelleted, fixed with buffered formalin for 24 hours,and embedded in paraffin. Tissue sections from normal kidneywere also used because the reactivity of proximal tubules differsfrom that of glomeruli, so that the results of P-glycoproteinimmunostaining are positive in proximal tubules and negativein glomeruli.24,25 The optimal dilutions were found to be 1:3for C219, 1:80 for MRK16, and 1:20 for JSB-1. Only JSB-1 andMRK16 were used in the tumor samples because these producedthe least variation in the results of immunostaining in differentexperiments and produced uniform results in tissue sections.
Five-micrometer sections from undecalcified, formalin-fixed,paraffin-embedded tissue samples were placed on poly-l-lysine–coatedslides (Sigma, St. Louis). The avidin–biotin–peroxidaseprocedure was used for immunostaining.26 After deparaffinizationand rehydration, the sections were treated with 0.3 percentmethanol–hydrogen peroxide to block endogenous peroxidaseactivity, incubated with normal horse serum (Vector, Burlingame,Calif.) for 30 minutes at 37°C, and incubated with primaryantibody overnight in a moist chamber at 4°C. The followingday, the tissue sections were incubated with secondary biotinylatedhorse antimouse antibody and with avidin–biotin–peroxidasecomplex (Vector). The final reaction product was revealed byexposure to 0.03 percent diaminobenzidine (Sigma), and the nucleiwere counterstained with Gill's hematoxylin. For each specimen,a negative control was obtained by staining the sample withthe secondary antibody only, and a positive control was obtainedby using a monoclonal antibody for vimentin (V-9, Boehringer,Mannheim, Germany; 1:100 dilution) as a primary antibody. Ineach experiment, normal kidney tissue was also included as acontrol.
The results of P-glycoprotein immunostaining were independentlyinterpreted by three observers who had no previous knowledgeof the clinical outcome in each patient. The results were expressedaccording to a semiquantitative scale. Tumor samples were gradedas zero when there was a complete absence of staining for P-glycoprotein.The P-glycoprotein–positive tumor samples were gradedfrom 1 to 3 according to the distribution of positivity andthe degree of immunostaining of the plasma membrane and theGolgi region, as follows: a score of 1, scattered positive cells(involvement of less than 10 percent of the specimen) and weakimmunostaining; a score of 2, diffuse positivity (more than10 percent of the specimen) and weak immunostaining; and a scoreof 3, diffuse positivity (more than 10 percent of the specimen)and strong immunostaining (Figure 1A, Figure 1B, Figure 1C,and Figure 1D). The highest degree of positivity found in anyarea of the section was recorded. On the basis of the findingsobtained in osteosarcoma cell lines, increased levels of P-glycoproteinwere inferred in the samples graded either 2 or 3.
Figure 1. Immunohistochemical Detection of P-Glycoprotein in Osteosarcoma with the Monoclonal Antibody JSB-1.
The brown stain indicates the presence of P-glycoprotein in the tumor cells. There is no detectable staining in Panel A, only a few positive cells (less than 10 percent) in Panel B (arrows), weak reactivity in the majority of cells in Panel C, and intense staining in the majority of cells in Panel D (all panels, x500). Tumors with immunostaining of more than 10 percent of cells (Panels C and D) were considered to be positive for P-glycoprotein.
Statistical Analysis
Fisher's exact test27,28 was used to evaluate the associationbetween two dichotomous variables. Kaplan–Meier plots29and the log-rank test30 were used to evaluate the associationof the percentage of necrosis and the expression of P-glycoproteinwith event-free survival. Cox proportional-hazards regressionanalysis with forward selection of variables31 was performedto estimate the rate ratios for possible risk factors for theoccurrence of adverse events. Data analysis was performed withthe SAS/PC statistical package, version 6.04 (SAS Institute,Cary, N.C.).
Results
Of the 92 osteosarcomas we examined, 56 did not show any immunoreactivityfor P-glycoprotein and 8 had only scattered positive cells.These 64 tumors (70 percent) were considered P-glycoprotein–negative.Of the remaining 28 tumors, P-glycoprotein immunostaining wasdiffuse, although weak in intensity, in 12, and diffuse andstrong in 16. These 28 tumors (30 percent) were considered tohave increased levels of P-glycoprotein. In these specimens,both membrane and cytoplasmic immunoreactivity was observedon exposure to the monoclonal antibodies JSB-1 and MRK16.
Association of the Expression of P-Glycoprotein with Clinical and Pathological Features
Immunostaining for P-glycoprotein was positive more frequentlyin female patients than in male patients, but this associationwas not statistically significant. In contrast, increased levelsof P-glycoprotein were more frequent in patients 14 years oldor younger than in older patients. Elevated levels of P-glycoproteinwere also associated with the anatomical site; proximal tumors,arising in the pelvis (6 patients), proximal femur (5 patients),or proximal humerus (15 patients) were more frequently positivefor P-glycoprotein than distal tumors, arising in the distalfemur (39 patients), tibia (25 patients), distal humerus (1patient), or radius (1 patient). No significant difference inthe incidence of P-glycoprotein positivity was found among thehistologic subtypes. However, P-glycoprotein levels were notelevated in grade 3 tumors, but were elevated in one third ofgrade 4 osteosarcomas. The level of expression of P-glycoproteindid not differ significantly between patients in whom resectionwas adequate (radical or wide margins) and those in whom resectionwas inadequate (marginal or intralesional margins).
No relation was found between the level of expression of P-glycoproteinand the extent of tumor necrosis after preoperative chemotherapy;overexpression of P-glycoprotein was found in tumors from 6of 25 patients (24 percent) with a poor response (<90 percenttumor necrosis) and from 22 of 67 patients (33 percent) witha good response (>90 percent tumor necrosis) (P = 0.46).
The actuarial event-free survival of the subgroup of 92 patientsthat was the focus of this study was nearly identical to thatof the entire group of 164 patients (P = 0.45). In the subgroup,increased levels of P-glycoprotein were significantly associatedwith a decreased probability of remaining event-free after diagnosis(P = 0.002) (Figure 2); the probability of remaining event-freewas 42 percent in patients with increased levels of P-glycoproteinand 80 percent in patients without such overexpression. By contrast,the degree of necrosis (>90 percent or <90 percent) afterpreoperative chemotherapy in the subgroup of 92 patients wasnot significantly related to the probability of remaining event-free(P = 0.09), in agreement with the findings in the entire groupof 164 patients (Figure 3).15 Other clinicopathological features,including age, sex, anatomical site, histologic grade and subtype,and surgical margin, were not associated with the outcome (datanot shown).
Figure 3. Probability of Event-free Survival in 92 Patients with Osteosarcoma and either Good or Poor Histologic Responses to Preoperative Chemotherapy.
The response to chemotherapy was considered good if the extent of tumor necrosis was 90 percent or greater and poor if it was less than 90 percent. Each tick mark represents a patient who had not had any adverse events as of the time of the last follow-up visit.
Table 2 shows the results of Cox proportional-hazards regressionanalysis in which the response to P-glycoprotein immunostainingand the histologic response after preoperative chemotherapywere adjusted simultaneously to estimate the rate ratios forthe occurrence of adverse events in patients with osteosarcoma.After adjustment for possible confounders, positive immunostainingfor P-glycoprotein persisted as an independent risk factor fora poor outcome. Patients with increased levels of P-glycoproteinhad a significantly higher rate ratio for adverse events (3.37)than patients without such overexpression. Multivariate analysisrevealed that a poor response to chemotherapy was also associatedwith a higher probability of adverse events.
Table 2. Results of Cox Proportional-Hazards Regression Analysis, Adjusted for the Response to P-Glycoprotein Immunostaining and the Histologic Response after Pre-operative Chemotherapy.
Discussion
We found that high levels of P-glycoprotein in tumor cells wereprognostic in patients with osteosarcoma. This highly malignantbone tumor commonly affects adolescents and young adults and,when treated only with surgical removal of the primary lesion,has an extremely aggressive course, with the appearance of metastasesin more than 80 percent of cases.32 The addition of chemotherapyto a regimen of surgery has significantly improved the outcomeof osteosarcoma,6,15,33 but despite an increase in the long-termrate of disease-free survival, metastases develop in a considerableproportion of patients, most of whom die. Since resistance toantineoplastic agents is likely to be a critical factor in theprogression of the disease, an understanding of the mechanismsof refractoriness to chemotherapy is important. Multidrug resistance,a well-characterized type of resistance mediated by the mdr1gene product P-glycoprotein, involves, among other agents, doxorubicin,the most effective drug in the treatment of osteosarcomas.34
We used immunohistochemical methods to examine paraffin-embeddedtissue sections of osteosarcoma for the presence of P-glycoprotein.We standardized the approach by using U-2 OS human osteosarcomacells and their multidrug-resistant derivative, U-2 OS/DX30,as negative and positive controls, respectively. The U-2 OS/DX30cell line is 15 times more resistant to doxorubicin than itsparent, U-2 OS.22 U-2 OS/DX30 has a low level of resistance,as is common in most human tumors.23 The reliability of themonoclonal antibodies against P-glycoprotein that we used (JSB-1and MRK16) has been extensively investigated in cell lines20,21,35and tissue samples.24,25,35,36 With the use of these antibodies,we were able to evaluate immunohistochemically how representativea specimen was as well as its P-glycoprotein status. Moreover,the availability of archival specimens for this analysis enabledus to evaluate the usefulness of P-glycoprotein status as aprognostic marker after an adequate clinical follow-up period.
Reports of multidrug resistance in sarcomas suggest that levelsof P-glycoprotein may be increased at the onset of clinicaldisease,37,38,39,40,41 and in soft-tissue sarcomas of childhood,a finding of increased levels of P-glycoprotein is stronglyassociated with a poor outcome.42 In our study of patients withhigh-grade osteosarcoma of the extremities, all of whom weretreated with the same regimen of chemotherapy in addition tosurgical removal of the primary lesion, increased levels ofP-glycoprotein were significantly associated with a poorer event-free–survivalrate.
Our results indicate that immunoreactivity to P-glycoproteinand, to a lesser extent, chemotherapy-induced necrosis are twovariables related to the clinical outcome of high-grade osteosarcoma.The histologic response to chemotherapy is generally consideredthe most reliable indicator of the clinical course of osteosarcoma,43although, at least by univariate analysis, its prognostic valueappears to be consistently reduced in the long term.15
The absence of a relation between the level of P-glycoproteinand the extent of tumor necrosis might reflect the killing ofa majority of tumor cells by a drug that acts independentlyof P-glycoprotein. It is also possible that P-glycoprotein statusand tumor necrosis status identify different phenomena44; increasedlevels of P-glycoprotein may represent not only resistance tochemotherapy, but also tumor progression.
The most important finding of this study is that in patientswith high-grade osteosarcomas of the extremities, which hadnot metastasized at the beginning of the study, the presenceof increased levels of P-glycoprotein could be used to identifytumors with a tendency to progress despite chemotherapy. Therisk of adverse events was increased more than threefold inpatients whose tumors had increased levels of P-glycoprotein.Moreover, in patients whose tumors contained little or no P-glycoprotein,the probability of event-free survival was significantly higherthan in patients whose tumors had increased levels of P-glycoprotein,regardless of the histologic response to chemotherapy. Anotheradvantage of the use of P-glycoprotein status over estimationof the extent of necrosis as a prognostic factor is that theformer can be determined at the time of initial diagnosis, beforeany therapy is initiated.
Our results may influence the selection of patients for newtreatments for osteosarcoma that combine conventional antineoplasticdrugs and agents that can modulate the multidrug-resistancephenotype.10,11,13,45 Agents that modify the multidrug-resistancephenotype, such as verapamil and cyclosporine, completely reversethe phenotype in vitro by competing with anticancer drugs forbinding sites on P-glycoprotein,46,47 and they have been includedin recent protocols for a number of malignant conditions.12,13,14,45,48Patients with osteosarcoma and increased levels of P-glycoproteinmay benefit from the addition of such agents to the treatmentregimen to enhance the cytotoxic effects of chemotherapy.
Supported by grants from the Istituti Ortopedici Rizzoli (RicercaCorrente Progetto 95005, and Ricerca Finalizzata Studio deiMeccanismi Coinvolti nella Farmacoresistenza e Validazione diIndicatori della Chemiosensibilità nei Sarcomi Muscolo-scheletrici)and the Associazione Italiana per la Ricerca sul Cancro.
Source Information
From the Dipartimento di Oncologia, Istituti Ortopedici Rizzoli, Bologna, Italy.
Address reprint requests to Dr. Baldini at the Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, 40136 Bologna, Italy.
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