|
Advertisement |
|
|
||
 |
|
|||||
|
|||||
|
 |
||||||
|
||||||
|
||||||
|
© 2003 American Society for Clinical Oncology Value of P-Glycoprotein and Clinicopathologic Factors as the Basis for New Treatment Strategies in High-Grade Osteosarcoma of the Extremities
From the Laboratorio di Ricerca Oncologica, Sezione di Chemioterapia, Servizio di Anatomia Patologica, Va Divisione, and Unita’ di Chirurgia Generale, Istituti Ortopedici Rizzoli, Bologna, Italy. Address reprint requests to Massimo Serra, Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, 40136 Bologna, Italy; email: massimo.serra{at}ior.it.
Purpose: To evaluate the prognostic value of P-glycoprotein and clinicopathologic parameters in a large series of high-grade osteosarcoma (OS) patients treated at the Rizzoli Institute. Patients and Methods: With the use of immunohistochemistry, P-glycoprotein was assessed in 149 patients with primary, nonmetastatic, high-grade OS who were homogeneously treated with chemotherapy protocols based on doxorubicin, high-dose methotrexate, and cisplatin and the addition of ifosfamide in the postoperative phase. Results: P-glycoprotein positivity was found in 47 of 149 cases (32%) and was significantly associated with a higher incidence of relapse and a worse outcome, as was age younger than 12 years and tumor volume greater then 150 mL at diagnosis. Multivariate analysis further confirmed the prognostic value of these parameters, which all were independent adverse prognostic factors. Event-free survival and proportional hazards regression analyses confirmed that overexpression of P-glycoprotein at clinical onset is the most important adverse prognostic factor for high-grade OS patients treated with these chemotherapy protocols. Conclusion: Increased P-glycoprotein levels, together with tumor volume and age, should be taken into consideration to identify, at time of diagnosis, subgroups of OS patients with a higher risk of recurrence. This subgroup identification will constitute the basis for drawing individualized treatment protocols on the basis of risk evaluation, with the aim of using more aggressive chemotherapy, or combination chemotherapy with other adjuvants, only in those patients for which more aggressive regimens are strictly necessary and warranted.
OSTEOSARCOMA (OS), the most common malignant tumor of bone, can appear in distinct clinical forms with different degrees of malignancy and prognosis.1 In particular, high-grade OS is characterized by a natural history of rapid growth and early development of distant metastases. Even in the most favorable subgroup of OS with high-grade features (the so-called "classical OS"), which includes patients younger than 40 years with tumors localized in the extremities and without any evidence of metastases at diagnosis, the survival rate does not exceed 60% to 70%.2–5 During the past three decades, long-term prognosis of high-grade OS patients has significantly improved, mainly because of the introduction of adjuvant and neoadjuvant chemotherapy, which has resulted in better control of both local growth and metastatic spread of tumor cells.6–8 Accordingly, clinical data have demonstrated that acquired resistance to chemotherapy is the major obstacle to successful treatment of OS and is associated with a higher risk of relapse and a worse outcome.2,7,9,10 In the last 10 years, attempts to change or further intensify chemotherapeutic regimens with the aim of increasing response to drug treatments have not significantly improved the outcome of OS patients, demonstrating that the current treatment regimens have most probably reached an impasse that is difficult to overcome.7,10,11 This evidence supports the need to identify predictive markers that may be used to determine, at presentation, patients who are likely to respond to conventional treatments and who, therefore, may be considered for stratified therapies on the basis of individual risk evaluation. Several sometimes contradictory findings have been reported regarding the factors that are associated with an increased risk of adverse events and unfavorable clinical outcome in high-grade OS. Among these, the extent of tumor necrosis after preoperative chemotherapy,12 tumor volume at diagnosis,5,13 pediatric age,5 overexpression of P-glycoprotein14,15 with a consequent development of drug resistance,16,17 and expression of the HER2/erbB-2 gene,18,19 are the factors most frequently reported to be associated with a worse clinical outcome of high-grade OS patients who are treated with surgery and multiagent chemotherapy. However, because of the lack of a general consensus regarding their prognostic value, most of these factors are rarely taken into consideration for designing treatment regimens, and none is currently used to stratify patients before the start of therapy. Moreover, it is still not clear whether these parameters need to be considered as independent adverse prognostic markers, or whether they may act jointly in determining a reduced response rate to chemotherapy, which eventually leads to a worse outcome. In this study, we assessed the expression of P-glycoprotein in a large, retrospective series of high-grade OS patients, homogeneously treated at the Rizzoli Institute (Istituti Ortopedici Rizzoli) in Bologna, Italy, with neoadjuvant chemotherapy protocols based on doxorubicin, high-dose methotrexate, and cisplatin, with the addition of ifosfamide in the postoperative phase. We analyzed P-glycoprotein expression in relation to other clinicopathologic features and clinical outcome. The final end point of this retrospective analysis was to identify those markers that may be useful in stratifying, at diagnosis, high-grade OS patients into high- and low-risk subgroups to plan new treatment protocols.
Patient Eligibility and Treatment Protocols Between September 1986 and December 1992, 300 patients with newly diagnosed OS were seen at the Rizzoli Institute and entered on the IOR/OS-2, IOR/OS-3a, or IOR/OS-3b chemotherapy protocols. The inclusion criteria for these chemotherapy protocols were high-grade OS of the extremity, age younger than 40 years, absence of metastases at the time of diagnosis, and no prior chemotherapy or surgical treatment for bone lesions. All patients underwent the neoadjuvant chemotherapy protocols specified above, which were basically the same regimen with minor modifications. As reported in detail in previous publications,4,20,21 preoperative chemotherapy was based on two cycles of high-dose methotrexate (8 g/m2 in IOR/OS-2, 10 g/m2 in IOR/OS-3a, and 12 g/m2 in IOR/OS-3b) followed by a combination of cisplatin 120 mg/m2 and doxorubicin 60 mg/m2. On the basis of the evaluation of tumor necrosis after preoperative chemotherapy, a good histologic response was considered when the extent of tumor necrosis was 90% or greater. Postoperative chemotherapy used the same drugs of the preoperative phase, with the addition of ifosfamide 10 g/m2 for patients with a poor histologic response in IOR/OS-2 and IOR/OS-3a, or for all patients in the IOR/OS-3b protocol. After the end of postoperative chemotherapy, patients were continuously followed and clinical data were continuously updated. Adverse events were defined as recurrence of the tumor at any site or death during remission, and event-free survival was calculated from the date of initial diagnosis. Tumor volume was evaluated on computed tomography scans taken at the time of diagnosis, as previously described.5 According to volume, tumors were classified into two groups by using the value of 150 mL as a cutoff, as suggested by the Cooperative Osteosarcoma Study Group (COSS) group study.13
Immunohistochemistry Expression of P-glycoprotein was evaluated with three monoclonal antibodies, which react with different, mutually exclusive epitopes of this protein. The antibodies used were JSB-1 (Sanbio, Uden, the Netherlands), MRK16 (Kamiya Biomedical, Thousand Oaks, CA) and C494 (Signet Laboratories, Dedham, MA). Immunohistochemistry was performed on 4- to 6-µm sections obtained from paraffin-embedded tumor specimens by using an avidin-biotin peroxidase complex method (Vectastain ABC kit, Vector Laboratories, Burlingame, CA), as previously described.14,16 Tissue sections were dewaxed with xylene for 60 minutes and gradually rehydrated with ethanol and water. After blocking of endogenous peroxidase activity by treatment with methanol/1% hydrogen peroxide for 30 minutes, sections were incubated with normal horse serum (Vector Laboratories) for 30 minutes at room temperature. Primary antibodies were incubated overnight at 4°C at the following concentrations: 12.5 µg/mL for JSB-1, 0.003 µg/mL for MRK16, and 0.2 µg/mL for C494. After washing, sections were subsequently incubated for 30 minutes at room temperature with a biotinylated horse antimouse antibody and with an avidin-biotin peroxidase complex (Vector Laboratories). The final reaction product was revealed by incubation with diaminobenzidine (Sigma Chemical, St. Louis, MO), and nuclei were counterstained with Gill’s hematoxylin. For each specimen, both negative and positive controls for immunostaining were performed. Negative controls were carried out by replacing the primary antibody with normal horse serum. As a positive control for P-glycoprotein, sections of normal kidney were used because of the reported overexpression of this protein in proximal tubuli.22 In addition, a positive control for the antigenicity of the tumor specimen was also included by incubating one section with the V9 antivimentin monoclonal antibody (Roche Molecular Biochemicals, Mannheim, Germany). Samples were scored separately by two independent investigators and only specimens with a diffused immunostaining for P-glycoprotein were considered positive. Determination of cases that showed discrepancy between the three anti-P-glycoprotein antibodies were repeated twice, and cases were classified on the basis of the most frequently recurring degree of immunostaining. In this study, immunohistochemistry was performed on 157 cases, eight of which were finally excluded for the lack of any positive reaction to vimentin.
Statistics
The group of 149 patients included in this study was representative of the whole series of 300 patients who underwent the three chemotherapy protocols considered here. In fact, as shown in Table 1  , no significant differences were found between these two groups of patients. All the clinicopathologic features presented by these groups of patients closely reflect the incidence and the distribution of these parameters in classical OS.1
Expression of P-Glycoprotein and Association With Clinicopathologic Features Of the 149 OS cases included in this study, 47 were P-glycoprotein-positive (32%), and 102 were P-glycoprotein-negative (68%). P-glycoprotein positivity was not significantly associated with any clinicopathologic feature (Table 2 ). However, there was a clear trend toward a higher frequency of P-glycoprotein positivity in patients age 12 years or younger compared with older patients.
No significant differences were found concerning the incidence of P-glycoprotein positivity in patients treated with the three chemotherapy protocols considered in this study (Table 2  ).
Considering the parameters associated with response to chemotherapy, no relationship was found between the expression of P-glycoprotein and the histologic response to preoperative chemotherapy evaluated as an extent of tumor necrosis. In fact, the incidence of P-glycoprotein-positive cases was similar in the subgroups of poor and good responders (Table 2
Clinical Outcome and Survival Analysis
Univariate analysis demonstrated that a higher relapse rate was significantly associated with P-glycoprotein positivity, age younger than 12 years, and tumor volume at diagnosis being greater than 150 mL (Table 3
The event-free survival curve of the 149 patients considered here showed a 63.1% survival rate (Fig 1 ), similar to the previously reported 58.8% survival probability of the whole group of 300 patients.5 Moreover, no differences were found among the three chemotherapy protocols, which showed similar event-free survival curves (Fig 1).
The analysis of event-free survival curves confirmed that only P-glycoprotein positivity, age younger than 12 years, and tumor volume at diagnosis being greater than 150 mL were significantly associated with a higher probability of adverse events and a worse outcome (Fig 2 ). In all the subgroups of patients, the majority of adverse events occurred between the second and fifth year of follow-up (Fig 2).
Multivariate analysis was performed for all the parameters that were significantly associated with a worse clinical outcome by the univariate approach. The Cox proportional hazards regression analysis further confirmed that P-glycoprotein positivity, age younger than 12 years, and tumor volume at diagnosis being greater than 150 mL were all independent risk factors associated with a poor outcome. In particular, the fold-increase in rate ratio for adverse events was 3.4 for P-glycoprotein positivity, 2.7 for age younger than 12 years, and 2.1 for tumor volume at diagnosis being greater than 150 mL (Table 4 ).
No relationships were found between these different parameters, indicating that they act independently in determining an adverse prognosis. However, by grouping patients according to these three markers (P-glycoprotein-negative/age >12 years/tumor volume  150 mL v P-glycoprotein-positive/age 12 years/tumor volume > 150 mL), it appeared that those patients who present simultaneously these three adverse prognostic factors at diagnosis have a high probability of early relapse (within the second year of follow-up) and a low survival rate (Fig 3).
The majority of patients with classical OS (patients younger than 40 years with tumors localized in the extremities and without any evidence of metastases at diagnosis) are currently successfully treated with standard regimens of multidrug, neoadjuvant chemotherapy. However, 30% to 40% of these patients still relapse, and many of them die from progression of the disease.3–5,7 Clinical studies have clearly demonstrated that response to chemotherapy is a critical factor, which can dramatically influence the clinical outcome of high-grade OS patients.6–8,11 However, attempts to intensify the standard chemotherapeutic regimens have resulted in a general increase of the collateral toxicity of anticancer agents, without any further significant improvement in the outcome of OS patients.7,8,11 This evidence demonstrates that current treatments for high-grade OS have most probably reached a survival plateau level and that individualized regimens, on the basis of individual risk evaluation, are necessary to improve the clinical results. Several different clinical and experimental studies have focused on the analysis of factors that may be predictive for prognosis of high-grade OS patients, but the difference in type and number of variables considered, together with the different characteristics of patients and treatment regimens, have resulted in reports with uncertain and sometimes conflicting data.7,8 Moreover, it has to be taken into account that the predictive value of prognostic factors is influenced not only by the inherent biology of the tumor but also by treatment modalities, and therefore, factors that predict outcome for one regimen may not be valid for alternative protocols. A number of different factors associated with worse prognosis have been identified in recent years, but there still is no general consensus about their relative prognostic value. These prognostic markers include tumor necrosis after preoperative chemotherapy,12 tumor volume at diagnosis,5,13 pediatric age,5 and expression of P-glycoprotein9,10,14–17,23 or HER2/erbB-2.18,19 In particular, the adverse prognostic value of P-glycoprotein has been clearly demonstrated to be associated with lack of responsiveness to cytotoxic drugs and not to an increased biologic aggressiveness of P-glycoprotein–positive OS cells.24,25 Although the adverse prognostic value of increased levels of P-glycoprotein expression at the time of diagnosis has been confirmed in several studies, performed by different institutions, a few data recently questioned the clinical relevance of this marker.19,26 In any case, no clear data have been reported so far about the possible relationship between P-glycoprotein expression and other clinicopathologic characteristics, mostly because of the insufficient number of patients included in the studies, heterogeneity of treatments, and short follow-up. This is the first report in which the correlation between P-glycoprotein expression and clinicopathologic parameters is investigated in a large series of OS patients with a long follow-up, in the context of homogeneous neoadjuvant chemotherapy regimens, mainly based on doxorubicin, high-dose methotrexate, and cisplatin. A previous clinical evaluation of the whole group of 300 patients who underwent these three chemotherapy protocols demonstrated that the factors significantly associated with a shorter disease-free survival were tumor volume at diagnosis more than 150 mL, age younger than 12 years, and osteoblastic or chondroblastic histologic subtypes.5 This study demonstrates, in agreement with the majority of previous findings, that overexpression of P-glycoprotein at the time of diagnosis is the most important adverse prognostic factor in patients with high-grade OS of the extremities treated with these neoadjuvant chemotherapy protocols. The prognostic value of tumor volume and age was confirmed, although no relationship was found between the three parameters identified as predictive factors by the univariate and multivariate analyses. Despite the fact that these three prognostic factors are independent from one another, when considered together, they were capable of identifying subgroups of OS patients with high or low survival probabilities. In contrast, no evidence of predictive value for histologic subtype, which was reported in the study of Ferrari et al,5 emerges from this study, most probably because of the small number of patients with nonosteoblastic/nonchondroblastic tumors. Particular attention must be addressed to tumor necrosis, which in different studies has been described as an important prognostic factor in high-grade OS.12,27–30 In this study, poor tumor necrosis after preoperative chemotherapy was not significantly associated either with a higher relapse rate or with a worse outcome. For the 138 patients treated according to IOR/OS-2 and IOR/OS-3a protocols, this may be partially explained by the fact that all patients with a poor response to preoperative chemotherapy received ifosfamide in the postoperative phase. The addition of this drug could have positively influenced the prognosis of poor responders by consequently decreasing the prognostic significance of tumor necrosis. For protocol IOR/OS-3b, the small sample size of 11 patients who received ifosfamide in the postoperative chemotherapy independently from the amount of tumor necrosis after the preoperative phase does not allow a complete explanation of this phenomenon. As previously reported,14,16,31 no relationship was found between the level of P-glycoprotein expression and tumor necrosis after preoperative chemotherapy. The absence of a relation between these two parameters may be explained by the fact that, most probably, P-glycoprotein status and tumor necrosis identify different, not completely overlapping phenomena related to the response to chemotherapy. In fact, tumor necrosis is the result of cumulative response to all drugs administered, including those that act independent from P-glycoprotein levels and activity. Recent revisions of clinical data have suggested that, most likely, the majority of patients with classical OS could be efficiently treated with optimal doses of only the most active agents for treatment of this tumor (doxorubicin, methotrexate, and cisplatin). In fact, the optimal use of these agents has been demonstrated to be able to reach the survival rate of other, more toxic, multidrug regimens.7,25,32,33 Therefore, the possibility of identifying, at clinical onset, high-risk groups of patients by taking into consideration reliable prognostic markers could be the basis for administering the most toxic treatments, involving additional anticancer agents, only to such patients for whom more aggressive regimens are strictly necessary and warranted. Our findings, by confirming the adverse prognostic value of increased P-glycoprotein levels, large tumor volume, and pediatric age, indicate that these parameters may serve to reach the goal of risk-adapted, individualized therapeutic regimens for high-grade OS patients.
We acknowledge Patrizia Bersani for assistance in performing tissue sections and Alba Balladelli, for correcting the spelling in this manuscript.
Supported by grants from the Istituti Ortopedici Rizzoli (Ricerca Corrente), Associazione Italiana per la Ricerca sul Cancro (AIRC), Italian Ministry of Health (Ricerca Finalizzata), and Italian Ministry for Education, University, and Research (MURST).
1. Campanacci M: Bone and Soft Tissue Tumors. Wien, Springer, 1999, pp 463–491 2. Souhami RL, Craft AW, Van der Eijken JW, et al: Randomised trial of two regimens of chemotherapy in operable osteosarcoma: A study of the European Osteosarcoma Intergroup. Lancet 350:911–917, 1997[CrossRef][Medline] 3. Meyers PA, Heller G, Healy J, et al: Chemotherapy for nonmetastatic osteogenic sarcoma: The Memorial Sloan-Kettering experience. J Clin Oncol 10:5–15, 1992[Abstract] 4. Bacci G, Picci P, Ferrari S, et al: Primary chemotherapy and delayed surgery for nonmetastatic osteosarcoma of the extremities. Results in 164 patients preoperatively treated with high doses of methotrexate, followed by cisplatin and doxorubicin. Cancer 72:3227–3238, 1993[CrossRef][Medline]
5. Ferrari S, Bertoni F, Mercuri M, et al: Predictive factors of disease-free survival for non-metastatic osteosarcoma of the extremity: An analysis of 300 patients treated at the Rizzoli Institute. Ann Oncol 12:1145–1150, 2001 6. Goorin AM, Abelson HT, Frei III E: Osteosarcoma: Fifteen years later. N Engl J Med 313:1637–1643, 1985[Medline] 7. Bruland ØS, Pihl A: On the current management of osteosarcoma. A critical evaluation and a proposal for a modified treatment strategy. Eur J Cancer 33:1725–1731, 1997[CrossRef][Medline] 8. Bramwell VHC: Osteosarcomas and other cancers of bone. Curr Opin Oncol 12:330–336, 2000[CrossRef][Medline] 9. Baldini N, Scotlandi K, Serra M, et al: P-glycoprotein expression in osteosarcoma: A basis for risk-adapted adjuvant chemotherapy. J Orthop Res 17:629–632, 1999[CrossRef][Medline] 10. Bramwell VH: The role of chemotherapy in the management of non-metastatic operable extremity osteosarcoma. Semin Oncol 24:561–571, 1997[Medline]
11. Bacci G, Ferrari S, Bertoni F, et al: Long-term outcome for patients with nonmetastatic osteosarcoma of the extremity treated at the Istituto Ortopedico Rizzoli according to the Istituto Ortopedico Rizzoli/osteosarcoma-2 protocol: An updated report. J Clin Oncol 18:4016–4027, 2000 12. Davis AM, Bell RS, Goodwin PJ: Prognostic factor in osteosarcoma: A critical review. J Clin Oncol 12:423–431, 1994[Abstract]
13. Bieling P, Rehan N, Winkler P, et al: Tumor size and prognosis in aggressively treated osteosarcoma. J Clin Oncol 14:848–858, 1996 14. Serra M, Maurici D, Scotlandi K, et al: Relationship between P-glycoprotein expression and p53 status in high-grade osteosarcoma. Int J Oncol 14:301–307, 1999[Medline] 15. Hornicek FJ, Gebhardt MC, Wolfe MW, et al: P-glycoprotein levels predict poor outcome in patients with osteosarcoma. Clin Orthop 373:11–17, 2000
16. Baldini N, Scotlandi K, Barbanti-Bròdano G, et al: Expression of P-glycoprotein in high-grade osteosarcomas in relation to clinical outcome. N Engl J Med 333:1380–1385, 1995
17. Chan HS, Grogan TM, Haddad G, et al: P-glycoprotein expression: Critical determinant in the response to osteosarcoma chemotherapy. J Natl Cancer Inst 89:1706–1715, 1997 18. Onda M, Matsuda S, Higaki S, et al: ErbB-2 expression is correlated with poor prognosis for patients with osteosarcoma. Cancer 77:71–78, 1996[CrossRef][Medline]
19. Gorlick R, Huvos AG, Heller G, et al: Expression of HER2/erbB-2 correlates with survival in osteosarcoma. J Clin Oncol 17:2781–2788, 1999 20. Ferrari S, Mercuri M, Picci P, et al: Nonmetastatic osteosarcoma of the extremity: Results of a neoadjuvant chemotherapy protocol (IOR/OS-3) with high-dose methotrexate, intraarterial or intravenous cisplatin, doxorubicin, and salvage chemotherapy based on histologic tumor response. Tumori 85:458–464, 1999[Medline] 21. Bacci G, Ferrari S, Longhi A, et al: Neoadjuvant chemotherapy for high-grade osteosarcoma of the extremities: Long-term results for patients treated according to the Rizzoli IOR/OS-3b protocol. J Chemother 13:93–99, 2001[Medline]
22. van der Valk P, van Kalken CK, Ketelaars H, et al: Distribution of multi-drug resistance-associated P-glycoprotein in normal and neoplastic human tissues: Analysis with 3 monoclonal antibodies recognizing different epitopes of the P-glycoprotein molecule. Ann Oncol 1:56–64, 1990 23. Park YB, Kim HS, Oh JH, et al: The co-expression of p53 protein and P-glycoprotein is correlated to a poor prognosis in osteosarcoma. Int Orthop 24:307–310, 2001[CrossRef][Medline]
24. Scotlandi K, Serra M, Nicoletti G, et al: Multidrug resistance and malignancy in human osteosarcoma. Cancer Res 56:2434–2439, 1996 25. Scotlandi K, Manara MC, Serra M, et al: The expression of P-glycoprotein is causally related to a less aggressive phenotype in human osteosarcoma cells. Oncogene 18:739–746, 1999[CrossRef][Medline] 26. Posl M, Amling M, Grahl K, et al: P-glycoprotein expression in high grade central osteosarcoma and normal bone cells. An immunohistochemical study. Gen Diagn Pathol 142:317–325, 1997[Medline]
27. Bramwell VHC, Burgers M, Sneath R, et al: A comparison of two short intensive adjuvant chemotherapy regimens in operable osteosarcoma of the limbs in children and young adults: The first study of the European Osteosarcoma Intergroup. J Clin Oncol 10:1579–1591, 1992
28. Provisor AJ, Ettinger LJ, Nachman JB, et al: Treatment of nonmetastatic osteosarcoma of the extremity with preoperative and postoperative chemotherapy: A report from the Children’s Cancer Group. J Clin Oncol 15:76–84, 1997
29. Fuchs N, Bielack SS, Epler D, et al: Long-term results of the co-operative German-Austrian-Swiss osteosarcoma study group’s protocol COSS-86 of intensive multidrug chemotherapy and surgery for osteosarcoma of the limbs. Ann Oncol 9:893–899, 1998 30. Saeter G, Wiebe T, Wiklund T, et al: Chemotherapy in osteosarcoma. The Scandinavian Sarcoma Group experience. Acta Orthop Scand 285:74–82, 1999 (suppl) 31. Serra M, Scotlandi K, Manara MC, et al: Analysis of P-glycoprotein expression in osteosarcoma. Eur J Cancer 31A:1998–2002, 1995[Medline] 32. Delepine N, Delepine G, Bacci G, et al: Influence of methotrexate dose intensity on outcome of patients with high grade osteogenic osteosarcoma. Analysis of literature. Cancer 78:2127–2135, 1996[CrossRef][Medline] 33. Himelstein BP: Osteosarcoma and other bone cancers. Curr Opin Oncol 10:326–333, 1998[Medline] Submitted March 28, 2002; accepted October 7, 2002.
This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|||||||||||
|
|||||||||||
|
|
Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
|
TOP
ABSTRACT
INTRODUCTION
