Figures
Abstract
Background
A variety of surgical procedures are now available for tissue reconstruction after osteosarcoma excision, and an important prognostic factor is the evaluation of response to chemotherapy using histology. Although tumor-bearing autografts are useful tools for reconstruction, re-use of the primary tumor may make it difficult to assess the histological response to chemotherapy, since the entire tumor cannot be analyzed. Here, we analyzed the prognostic value of the histological response in the patients who received frozen tumor-bearing autografts for reconstruction.
Method
Retrospective analysis of the medical records of 51 patients with high-grade osteosarcoma of the extremities was performed. All patients received reconstruction using frozen tumor-bearing autografts. Tumor necrosis was evaluated in extraskeletal masses and cancellous bone.
Results
Five-year overall survival of patients with good and poor response to chemotherapy was 82.9% and 46.4%, respectively (P = 0.044), and 5-year event-free survival was 57.7% and 36.0%, respectively (P = 0.329). Multivariate analysis revealed that a poor histological response to chemotherapy was a significant prognostic factor for overall survival (P = 0.033).
Citation: Miwa S, Takeuchi A, Ikeda H, Shirai T, Yamamoto N, Nishida H, et al. (2013) Prognostic Value of Histological Response to Chemotherapy in Osteosarcoma Patients Receiving Tumor-Bearing Frozen Autograft. PLoS ONE 8(8): e71362. https://doi.org/10.1371/journal.pone.0071362
Editor: Marta M. Alonso, University Hospital of Navarra, Spain
Received: April 21, 2013; Accepted: June 26, 2013; Published: August 15, 2013
Copyright: © 2013 Miwa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no funding or support to report.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Before the introduction of chemotherapy, patients with osteosarcoma had an extremely poor prognosis and limb amputation was the standard treatment. One of the purposes of chemotherapy is the eradication of metastatic lesions; however, it is difficult to eradicate all tumor cells when metastatic lesions are clinically detectable. Therefore, metastatic disease at initial diagnosis is one of the most important prognostic factors.
The introduction of chemotherapy markedly stimulated the development of surgical procedures for osteosarcoma. Although mega-prosthesis is currently the most widely used reconstruction procedure after tumor excision, allograft, autograft, allograft-prosthetic composites, autograft-prosthetic composites, distraction osteogenesis, and rotationplasty have also been performed for bone reconstruction [1]–[8]. There are various kinds of autografts including iliac bone, vascularized fibula, and tumor-bearing bone grafts. For use in reconstruction, tumor-bearing bone requires pre-treatment to eliminate viable tumor cells, such as autoclaving, pasteurization, irradiation, or freezing in liquid nitrogen. In our institute, frozen bone autografts have been used for reconstruction following malignant bone tumor excision since 1999 [6]. Reconstruction using tumor-bearing bone creates a problem with assessment of chemotherapeutic effects, which is important information required for evaluating the chemotherapy treatment plan including the number of courses and anticancer drug choice, surgical procedure, and surgical margins. Chemotherapeutic effects are commonly assessed by comparing the cellularity and tumor necrosis of a biopsy specimen and resected bone. While a large area of the tumor can be assessed in cases of amputation and mega-prosthesis, only a part of the tumor is available in cases of reconstruction using tumor-bearing bone. The latter is assumed to decrease the accuracy of assessments of chemotherapeutic effects; however, this has not been analyzed objectively. Here, we have analyzed the accuracy and prognostic value of tumor necrosis following reconstruction using frozen tumor-bearing autografts.
Methods
Patients
A total of 59 patients with osteosarcoma were treated with reconstruction using frozen tumor-bearing autografts between January 1999 and November 2011; patients with low-grade osteosarcoma, truncal site, or non-surgical patients were excluded. This study was approved by the Institutional Review Board of the Kanazawa University Graduate School of Medical Science, Kanazawa, Japan. Written informed consent was obtained from all patients and/or their family.
All patients received chemotherapy according to the K2 protocol [6], which was modified for each patient based on their general condition, past history of chemotherapy, renal and liver functions. Three courses of chemotherapy using cisplatin (120 mg/m2), doxorubicin (30 mg/m2×3 days) and caffeine (1.5 g/m2×3 days) were administered at 3-week intervals. The effects of chemotherapy were evaluated radiologically after three courses of chemotherapy. An additional two courses of chemotherapy were administered to good responders. Poor responders either underwent surgery immediately or were given other drugs such as ifosfamide or etoposide. After surgical treatment, patients received 3–6 additional courses of intravenous cisplatin, doxorubicin, and caffeine. The primary tumor, local recurrence, and metastasis were assessed clinically and radiologically examined using X-ray, CT, MRI, and bone scintigraphy.
Surgical Procedure
Frozen tumor-bearing autografts were obtained by tumor excision, curettage, freezing in liquid nitrogen, and used in reconstruction (Fig. 1) [7].
b. Curettage.
Soft tissues were removed from the excised tumor-bearing bone. After cancellous bone was curetted, excess water in the bone was removed by suction to prevent bone damage due to ice expansion during freezing. Curetted cancellous bone and extraskeletal masses were evaluated histologically.
Reconstruction
The massive bone and osteochondral defects following resection were reconstructed using frozen tumor-bearing autografts and instruments such as prostheses, intramedullary nails, and plates.
Histological Evaluation of Chemotherapeutic Effect
Histological responses to chemotherapy were assessed by comparing tumor biopsy and resected specimens, including cancellous bone, as well as extraskeletal masses not requiring reconstruction (Fig. 1). All the curetted tumors and the extraskeletal masses including largest cross-section were observed to assess the response to chemotherapy. Histologic gradings were based on the degree of necrosis: grade IV (100% necrosis), grade III (90–99% necrosis), grade II (50–89% necrosis), and grade I (0–49% necrosis) [9]. Grade III and IV were classified as good responses to chemotherapy; grade I and II were poor responses.
Statistical Analysis
We examined the prognostic significance of age (<40 years vs. ≥40 years), gender, histological subtype (osteoblastic type or others), metastatic disease at initial diagnosis, and histological response (grade I and II vs grade III and IV). Overall survival and event-free survival were calculated using the Kaplan–Meier method with log-rank test. Multivariate Cox proportional hazards regression analyses were used to identify independent predictors of overall survival and event-free survival, which were defined as the time from the initial diagnosis to death from any cause and time from the initial diagnosis to metastasis, local recurrence, or death from any cause. Statistical significance was defined as P<0.05; factors with P<0.4 in univariate analysis were included in multivariate Cox proportional hazards models. Statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University).
Results
Patients
A total of 51 osteosarcoma patients receiving frozen tumor-bearing autografts were retrospectively reviewed, consisting 28 male and 23 female patients with a mean age of 21.2 years (range, 5–69) (Table 1). The mean follow-up period was 44.6 months (range, 7–177). At initial diagnosis, 16 patients (31.4%) had clinically detectable metastases, while the rest did not. Histologically, 37 were osteoblastic type, 10 were chondroblastic type, 2 were fibrous type, and 2 were the other type. The 3-, 5-, and 10-year overall survival rate of the study patients was 82.8%, 66.4%, and 55.3%, respectively (Fig. 2). The 3-, 5-, and 10-year event-free survival rate was 52.8%, 49.0%, and 49.0%, respectively (Fig. 2). In histological evaluation of chemotherapeutic effects, 21 patients were grade IV, 15 patients were grade III, 13 patients were grade II, and 2 patients were grade I. On the basis of the Enneking’s surgical staging, 2 patietns were classified as stage IIA, 33 as stage IIB, and 16 as stage IIIB [10].
Overall Survival
Kaplan–Meier analysis revealed that absence of metastasis at initial diagnosis was significantly associated with good overall survival in the patients (P<0.001; Table 2). The 5-year overall survival of patients with or without metastases was 37.9% and 88.3%, respectively (Fig. 3a). Histological response was also significantly associated with good overall survival (P = 0.044; Table 2). The 5-year overall survival rate of poor responders (less than 90% necrosis) was 46.4% compared with 82.9% in good responders (90% or more necrosis) (Fig. 3b). There were no significant associations between overall survival and age, gender, or pathological tumor type.
a. Metastatic lesion. b. Histological response.
Multivariate analysis showed that metastatic disease at initial diagnosis was the most important independent predictor of overall survival (HR, 8.33; CI, 1.9–36.1; P = 0.005), followed by poor histological response (HR, 3.98; CI, 1.1–14.1; P = 0.033; Table 3).
Event Free Survival
The presence of metastases at initial diagnosis (P<0.001) correlated to a significant degree with event-free survival (Table 4): the 5-year event-free survival of metastatic patients was 0%, compared with 69.4% for non-metastatic patients. There were no associations between event-free survival and age, gender, pathological type, and histological response to chemotherapy.
Multivariate analysis showed that metastatic disease at initial diagnosis was the only important independent predictor of event-free survival (HR, 15.66; CI, 4.8–51.5; P<0.001; Table 5). Histologically, poor response (necrosis <90%) did not show significant correlation with event-free survival (HR, 1.72; CI, 0.7–4.1; P = 0.217).
Prognostic Value of Histological Response to Chemotherapy in Patients with or without Metastasis
The study patients were divided into the patients without metastasis and the patients with metastases, and the prognostic values of histological response to chemotherapy were evaluated in each group. Among the patients without metastasis, 5-year overall survival rates of good responders and poor responders were 88.9% and 70.0%, respectively (P = 0.274; Fig. 4a). Five-year event-free survival rates of good responders and poor responders were 82.9% and 49.1%, respectively (P = 0.174; Fig. 4a). Among the patients with metastases, 3-, 5-year overall survival rates of good responders were 68.8% and 55.0%, compared with 25.0% and 0% in poor responders (P = 0.042; Fig. 4b).
a. Patients without metastasis. b. Patients with metastases.
Discussion
The proportion of limb salvage surgery in the treatment of osteosarcoma has increased since the 1980s. Currently, there are a variety of options of reconstructive procedures, including osteoarticular bone grafts, intercalary bone graft, autograft- or allograft-prosthetic composites, and mega-prosthesis [1]–[8], [11]–[14]. Although mega-prosthesis is a useful and commonly used reconstructive tool, tumor-bearing autograft is also useful for the reconstruction of massive bone and osteochondral defects following the resection of osteosarcoma. Although allografts are difficult to obtain in Asian countries for religious reasons, tumor-bearing autografts are readily available and have no compatibility problems. Before being used for reconstructive surgery, bone grafts must undergo one of several pre-treatments, including pasteurization, autoclaving, irradiation, or freezing in liquid nitrogen; the latter is particularly advantageous owing to its simplicity and rapidity [7], [8]. Furthermore, freezing maintains tissue microstructure and tumor antigens, whereas pasteurization and irradiation cause protein degradation [15]. Consequently, frozen autografts are associated with good bone induction and conduction as well as activation of tumor immunity [16]–[18]. Although many techniques for re-using tumor-bearing bone need osteotomy, which may cause non-union, this disadvantage is not associated with pedicle freezing since this can be performed without osteotomy [7], [8].
Histological response to chemotherapy is one of the most important prognostic factors for patients with osteosarcoma [19], [20]. The histological response to chemotherapy usually influences post-operative treatment, including possible changes of anticancer drugs and indicating the need for additional treatments such as radiation therapy. Many experimental studies have been reported the use of drug-response assay. Histoculture drug response assay (HDRA), which cultures three-dimensional tumor tissue, highly correlates with histological response to chemotherapy [21], [22]. Furthermore, there are reports which describing that chemosensitivity determined by the HDRA seems to be a strong predictor of survival in patients with malignant tumors [23]–[25]. Although there are few reports describing the use of HDRA for osteosarcoma, HDRA would be complementary to evaluation of chemotherapeutic effects. On the other hand, we previously reported that the chemotherapeutic effects evaluated by 99mTc-MIBI scintigraphy (99mTc-MIBI) and combined radiological score (CRS) had significant correlation with histological response to chemotherapy [26], [27]. Furthermore, the chemotherapeutic effects evaluated by 99mTc-MIBI and CRS correlated with overall survival [28]. Although chemotherapeutic effects are important information to make the surgical treatment plan, it is impossible to assess histological response before tumor excision. These reports suggest that 99mTc-MIBI and CRS could be used as supplementary tool for assessment of chemotherapeutic effects before surgical treatment.
Histological responses to chemotherapy are commonly evaluated by comparing necrosis of the resected tumor with the biopsy specimen. In our institute, chemotherapeutic effects have been evaluated according to Huvos grading system, one of the most standard grading systems [9]. Many studies have shown that the histological response to preoperative chemotherapy is one of the most important predictors for clinical outcome of osteosarcoma [19], [29]–[34]. In most of these studies, less than 90% of chemotherapy induced tumor necrosis rate was graded as “poor”, and 90% or more tumor necrosis rate was defined as “good”. Compared with poor responders, good responders had significantly higher event-free survival [27], [29], [32] and/or overall survival [19], [29]–[34]. However, the use of 90% necrosis as a cutoff point in assessment of chemotherapeutic effect is controversial [35], [36]. Some studies showed that histological response was not associated with survival of osteosarcoma patients [37]–[39]. Li et al. reported that tumor necrosis grouped at 90% was not associated with OS and EFS, while patients with greater than 70% necrosis rate had significantly higher EFS than those with less than 70% [35]. Harting reported that patients with less than 50% necrosis rate had significantly poor OS [36]. In further study, several cutoff points of tumor necrosis should be analyzed to define the adequate cutoff point.
Tumor cellularity and the degree of necrosis due to chemotherapy are heterogeneous throughout the tumor. An unsubstantiated disadvantage of using tumor-bearing autografts is that the histological assessment of the small, pre-reconstruction sample from the graft may not be representative of the cellularity and necrosis of the tumor as a whole. Here, we have addressed this important unknown by analyzing the correlation between histological response and prognosis in osteosarcoma patients who underwent reconstructive surgery using frozen tumor-bearing autografts. We found that the histological response determined from the graft biopsy was an important prognostic factor in these patients, indicating that this analysis was adequate and representative of the whole tumor. Our study only included patients who received frozen autografts; therefore, the accuracy of assessing the chemotherapeutic effect in the patients who receive tumor-bearing autograft including irradiated bone, pasteurized bone, and autoclaved bone should be discussed. Our data indicate that histological response to chemotherapy is also a reliable and important prognostic factor in osteosarcoma patients who received tumor-bearing autografts for reconstruction.
There are several limitations in our study. First, the patient numbers of the present study was small because osteosarcoma has low incidence in the whole population (2–3/million/year) [40]. Secondly, we did not compare the prognosis and histological responses to chemotherapy of patients receiving tumor-bearing autografts vs. a control group. Although there were patients who received mega-prosthesis or limb amputation during the study period, and their histological responses to chemotherapy could have been assessed by total tumor specimens, these patients were not sufficiently similar to be used as controls, e.g., based on tumor size, invasion into nerve or vessels, all of which may influence their overall and event-free survival. Therefore, it is unclear whether assessing histological response to chemotherapy using a small sample is an accurate analysis of the entire tumor, which would require a prospective and/or direct comparison of the different patient groups. Further study with more patients should also be performed to evaluate the predictive value of the tumor necrosis in patients receiving tumor-bearing autograft.
Conclusions
Histological response to chemotherapy and metastasis at diagnosis were both significantly correlated with overall survival. The correlation between the histological response to chemotherapy and overall survival suggested its accuracy as a prognostic factor for patients receiving tumor-bearing autografts for tissue reconstruction.
Author Contributions
Conceived and designed the experiments: SM AT TS HT. Performed the experiments: SM AT HI TS NY HN KH YT HK KI HT. Analyzed the data: SM AT TS HT. Contributed reagents/materials/analysis tools: SM AAT HI TS. Wrote the paper: SM AT HT.
References
- 1. Chen WM, Chen TH, Huang CK, Lin CF, Shih LY, et al. (2000) Treatment of high-grade primary osteosarcoma in extremities: 15-year clinical experience in VGH-Taipei. J Orthop Surg 17: 11–16.
- 2. Chen WM, Chen TH, Huang CK, Chiang CC, Lo WH (2002) Treatment of malignant bone tumours by extracorporeally irradiated autograft-prosthetic composite arthoplasty. J Bone Joint Surg Br 84: 1156–1161.
- 3. Chen WM, Wu PK, Chen CF, Chung LH, Liu CL, et al. (2012) High-grade osteosarcoma treated with hemicortical resection and biological reconstruction. J Surg Oncol 105: 825–829.
- 4. Grimer RJ (2005) Surgical options for children with osteosarcoma. Lancet Oncol 6: 85–92.
- 5. Tsuchiya H, Tomita K, Minematsu K, Mori Y, Asada N, et al. (1997) Limb salvage using distraction osteogenesis. A classification of the technique. J Bone Joint Surg Br 79: 403–411.
- 6. Tsuchiya H, Tomita K, Mori Y, Asada N, Yamamoto N (1999) Marginal excision for osteosarcoma with caffeine assisted chemotherapy. Clin Orthop Relat Res 358: 27–35.
- 7. Tsuchiya H, Wan SL, Sakayama K, Yamamoto N, Nishida H, et al. (2005) Reconstruction using an autograft containing tumour treated by liquid nitrogen. J Bone Joint Surg Br 87: 218–225.
- 8. Tsuchiya H, Nishida H, Srisawat P, Shirai T, Hayashi K, et al. (2010) Pedicle frozen autograft reconstruction in malignant bone tumors. J Orthop Sci 15: 340–349.
- 9. Rosen G, Caparros B, Huvos AG, Kosloff C, Nirenberg A, et al. (1982) Preoperative chemotherapy for osteogenic osteosarcoma: selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy. Cancer 49: 1221–1230.
- 10. Enneking WF, Spanier SS, Goodman MA (1980) A system for the surgical staging of musculoskeletal sarcoma. Clin Orthop Relat Res 153: 106–120.
- 11. Tsuboyama T, Toguchida J, Kotoura Y, Kasahara K, Hiraoka M, et al. (2000) Intra-operative radiation therapy for osteosarcoma in the extremities. Int Orthop 24: 202–207.
- 12. Manabe J, Ahmed AR, Kawaguchi N, Matsumoto S, Kuroda H (2004) Pasteurized autologous bone graft in surgery for bone and soft tissue sarcoma. Clin Orthop Relat Res 419: 258–266.
- 13. Lauritzen C, Alberius P, Santanelli F, Vällfors B, Lilja J, et al. (1991) Repositioning of craniofacial tumorous bone after autoclaving. Scand J Plast Reconstr Surg Hand Surg 25: 161–165.
- 14. Ehara S, Nishida J, Shiraishi H, Tamakawa Y (2000) Pasteurized intercalary autogenous bone graft: radiographic and scintigrahic features. Skeletal Radiol 29: 335–339.
- 15. Takata M, Sugimoto N, Yamamoto N, Shirai T, Hayashi K, et al. (2011) Activity of bone morphogenetic protein-7 after treatment at various temperatures: freezing vs. pasteurization vs. allograft. Cryobiology 63: 235–239.
- 16. Nishida H, Tsuchiya H, Tomita K (2008) Re-implantation of tumour tissue treated by cryotreatment with liquid nitrogen induces anti-tumour activity against murine osteosarcoma. J Bone Joint Surg Br 90: 1249–1255.
- 17. Tanzawa Y, Tsuchiya H, Shirai T, Hayashi K, Yo Z, et al. (2009) Histological examination of frozen autograft treated by liquid nitrogen removed after implantation. J Orthop Sci 14: 761–768.
- 18. Tanzawa Y, Tsuchiya H, Yamamoto N, Sakayama K, Minato H, et al. (2008) Histological examination of frozen autograft treated by liquid nitrogen removed 6 years after implantation. J Orthop Sci 13: 259–264.
- 19. Bielack SS, Kempf-Bielack B, Delling G, Exner GU, Flege S, et al. (2002) Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols. J Clin Oncol 20: 776–790.
- 20. Davis AM, Bell RS, Goodwin PJ (1994) Prognostic factors in osteosarcoma: a critical review. J Clin Oncol 12: 423–431.
- 21. Furukawa T, Kubota T, Hoffman RM (1995) Clinical applications of the histoculture drug response assay. Clin Cancer Res 1: 305–311.
- 22.
Hoffman RM (2010) Histocultures and Their Use. Encyclopedia of Life Sciences. John Wiley and Sons, Ltd.: Chichester.
- 23. Kubota T, Sasano N, Abe O, Nakano I, Kawamura E, et al. (1995) Potential of the histoculture drug-response assay to contribute to cancer patient survival. Clin Cancer Res 1: 1537–1543.
- 24. Vescio RA, Redfern CH, Nelson TJ (1987) In vivo-like drug responses of human tumors growing in three-dimensional gel-supported primary culture. Proc Natl Acad Sci USA 84: 5029–5033.
- 25. Freeman AE, Hoffman RM (1986) In vivo-like growth of human tumors in vitro. Proc Natl Acad Sci USA 83: 2694–2698.
- 26. Miwa S, Shirai T, Taki J, Sumiya H, Nishida H, et al. (2011) Use of 99mTc-MIBI scintigraphy in the Evaluation of the Response to Chemotherapy for Osteosarcoma: Comparison with 201Tl scintigraphy and angiography. Int J Clin Oncol 16: 373–378.
- 27. Miwa S, Taki J, Yamamoto N, Shirai T, Nishida H, et al. (2012) A novel combined radiological method for evaluation of the response to chemotherapy for primary bone sarcoma. J Surg Oncol 106: 273–279.
- 28.
Miwa S, Takeuchi A, Shirai T, Taki J, Yamamoto N, et al. (in press) Prognostic value of radiological response to chemotherapy in patients with osteosarcoma. PLoS ONE (in press).
- 29. Bacci G, Longhi A, Versari M, Mercuri M, Briccoli A, et al. (2006) Prognostic factors for osteosarcoma of the extremity treated with neoadjuvant chemotherapy: 15-year experience in 789 patients treated at a single institution. Cancer 106: 1154–1161.
- 30. Ferrari S, Bacci G, Picci P, Mercuri M, Briccoli A, et al. (1997) Long-term follow-up and post-relapse survival in patients with nonmetastatic osteosarcoma of the extremity treated with neoadjuvant chemotherapy. Ann Oncol 8: 765–771.
- 31. Ford S, Saithna A, Grimer RJ, Picci P (2004) Comparison of the outcome of conventional osteosarcoma at two specialist international orthopaedic oncology centres. Sarcoma 8: 13–18.
- 32. Pakos EE, Nearchou AD, Grimer RJ, Koumoullis HD, Abudu A, et al. (2009) Prognostic factors and outcomes for osteosarcoma: an international collaboration. Eur J Cancer 45: 2367–2375.
- 33. Bacci G, Bertoni F, Longhi A, Ferrari S, Forni C, et al. (2003) Neoadjuvant chemotherapy for high-grade central osteosarcoma of the extremity. Histologic response to preoperative chemotherapy correlates with histologic subtype of the tumor. Cancer 97: 3068–3075.
- 34. Bacci G, Mercuri M, Longhi A, Ferrari S, Bertoni F, et al. (2005) Grade of chemotherapy-induced necrosis as a predictor of local and systemic control in 881 patients with non-metastatic osteosarcoma of the extremities treated with neoadjuvant chemotherapy in a single institution. Eur J Cancer 41: 2079–2085.
- 35. Li X, Ashana AO, Moretti VM, Lackman RD (2011) The relation of tumour necrosis and survival in patients with osteosarcoma. Int Orthop 35: 1847–1853.
- 36. Harting MT, Lally KP, Andrassy RJ, Vaporciyan AA, Cox CS Jr, et al. (2010) Age as a prognostic factor for patients with osteosarcoma: an analysis of 438 patients. J Cancer Res Clin Oncol 136: 561–570.
- 37. Bacci G, Briccoli A, Ferrari S, Longhi A, Mercuri M, et al. (2001) Neoadjuvant chemotherapy for osteosarcoma of the extremity: long-term results of the Rizzoli's 4th protocol. Eur J Cancer 37: 2030–2039.
- 38. Le Deley MC, Guinebretière JM, Gentet JC, Pacquement H, Pichon F, et al. (2007) SFOP OS94: a randomised trial comparing preoperative high-dose methotrexate plus doxorubicin to high-dose methotrexate plus etoposide and ifosfamide in osteosarcoma patients. Eur J Cancer 43: 752–761.
- 39. Lewis IJ, Nooij MA, Whelan J, Sydes MR, Grimer R, et al. (2007) Improvement in histologic response but not survival in osteosarcoma patients treated with intensified chemotherapy: a randomized phase III trial of the European Osteosarcoma Intergroup. J Natl Cancer Inst 99: 112–128.
- 40. Bielack SS, Machatschek JN, Flege S, Jurgens H (2004) Delaying surgery with chemotherapy for osteosarcoma of the extremities. Expert Opin Pharmacother 5: 1243–1256.