Poor Prognosis Osteosarcoma: New Therapeutic Approach

ORIGINAL ARTICLE Poor prognosis osteosarcoma: new therapeutic approach

F Fagioli1, E Biasin1, OM Mereuta1, M Muraro1, R Luksch2, S Ferrari3, M Aglietta4 and E Madon1

1Stem Cell Transplantation and Cellular Therapy Unit, Regina Margherita Children’s Hospital, Turin, Italy; 2Department of Pediatric Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori di Milano, Milan, Italy; 3Division of Chemotherapy, Istituti Ortopedici Rizzoli, University of Bologna, Bologna, Italy and 4Department of Clinical Oncology, IRCC, University of Turin, Turin, Italy

Over the past 30 years,a significant improvement in the prognosis of patients with multifocal osteosarcoma7–9 prognosis of localized osteosarcoma of the extremities or with axial metastases6 is the most severe. has been observed. Despite these results,approximately 30–40% of patients will relapse,mostly within the first 3 years from diagnosis. The prognosis of patients with Indications and risk factors recurrent disease or metastases at diagnosis is poor. To improve the survival in this patient population,several Among patients with osteosarcoma, the prognosis is severe attempts have been made. An increased dose intensity of in (a) poor responders to neoadjuvant chemotherapy, (b) chemotherapy induces short lasting remission but does not patients without the possibility of primary tumor resection, increase the survival. In the era of targeted therapy,few (c) patients with metastases at diagnosis, in particular bone drugs have been tested with dismal results. The use of metastases and (d) recurrent disease with unresectable biological agents endowed with immunomodulant activity metastases or more than two resectable lung nodules.4 (that is IL-2) or reduced-intensity allogeneic hemopoietic Because of its poor prognosis, this group of patients SCT has produced intriguing results that need further represents the target for new therapeutic strategies. confirmation. In this context,an ongoing study explores the antitumor activity of specific T-cytotoxic lymphocytes. Bone Marrow Transplantation (2008) 41, S131–S134; doi:10.1038/bmt.2008.71 Results Keywords: high-grade osteosarcoma; high-dose chemo- High-dose chemotherapy therapy; immunotherapy; cytotoxic lymphocytes The use of high-dose chemotherapy with subsequent hemopoietic PBSC infusion has been investigated in a few studies. Thirty-two patients with metastatic osteosarcoma were included in an Italian Sarcoma Group (ISG) study. Two courses of high-dose carboplatin and etoposide were Introduction followed by autologous stem cell rescue: the 3-year overall survival was 20% with a 3-year disease-free survival of The prognosis and the quality of life of localized 12%.10 Similar data were obtained by the Cooperative osteosarcoma patients greatly improved over the last 30 German–Austrian–Swiss Osteosarcoma Study Group by years with overall survival rates increasing from 10% using different drugs in the high-dose setting.11 1 with surgery alone, usually amputation, to 70% with the Importantly, in the first group of patients there was a introduction of neoadjuvant chemotherapy and limb- high remission rate, although with a low disease-free 2,3 sparing surgery. However, recurrent disease occurs in survival.10 Therefore, this approach might be used to approximately 30–40% of patients with non-metastatic induce remission, but different strategies, such as immuno- osteosarcoma of the extremities. Recurrence is most therapy, should be explored to maintain the remission. common in the lungs. The mean 5-year post-relapse survival rate is about 28%, but some groups of patients have a 0% overall survival.4 Patients with metastatic Radiometabolic treatment disease at diagnosis have a poor prognosis with a 5-year Samarium-153 ethylenediamine tetramethylene phosphonic event-free survival of 30%.5,6 Among this group, the acid (EDTMP), a radio-labeled phosphonate, was developed for palliative treatment of bone metastases with a standard palliative dose of 1 mCi/kg. Attempts have been made to improve its activity by Correspondence: Dr F Fagioli, Stem Cell Transplantation and Cellular Therapy Unit, Regina Margherita Children’s Hospital, Piazza Polonia increasing the dose. 12 94, Turin 10126, Italy. Franzius et al. reported the treatment of six patients E-mail: [email protected] with 4 mCi/kg: 36 months later, one patient was still free of Treatment of high-risk osteosarcoma F Fagioli et al S132 (1)Patients' DCs pulsed with (2)Patients' DCs (3) Osteosarcoma autologous apoptotic tumor pulsed with tumor cell lines cells cell line lysates MAGE HOS SJSA-1 A1- A2- SART-3 Ag HOS A3 Ag MG-63 SSX-2 80 Gy 60 Gy 30 Gy Ag BB CTL

+ IL-12 + IL-12,15 + IL-2,12,15 Patients' + IL-2,7 PBMCs/CD8+ + IL-7 + IL-2,7 ELIS POT assay phenotype cytokine release

Figure 1 Study design.

progression. Anderson et al.13 treated 30 patients with CTL clones (10X) 30 mCi/kg followed by PBSC support reporting no data on disease efﬁcacy. Minimal non-hematologic side effects were evidenced. We treated seven patients with poor prognosis osteosarcoma by administering samarium-153–EDTMO 10 mCi/kg and subsequent PBSCs. Five patients had progression of disease at a median of 2 months and died of disease at a median of 5 months after treatment. Two patients were still alive with stable disease at 7 and 12 months. Studies on the use of samarium-153–EDTMO are limited and not homogeneous. No conclusive data can be drawn, but in a new protocol of the ISG and AIEOP (Italian Association of Pediatric Hemato-Oncology), samarium-153–EDTMO is used after chemotherapy to consolidate the tumor response. CTL clones (40X)

Immunotherapy and non-myeloablative allo-SCT The IL-2 has been investigated as an immunotherapeutic approach on 18pediatric patients affected by osteosarcoma and treated with four courses of IL-2 and polychemother- apy. The results showed an immune activation in response to IL-2.14 Non-myeloablative allo-SCT is a form of immunotherapy linked to minor HLA antigens, which has been shown to be efﬁcient in different solid tumors.15–18 We reported the evidence of metastases regression in a 12-year-old boy with advanced osteosarcoma, in which the immunological anti-osteosarcoma effect was independent of the GvHD reaction.17 Goi et al.19 treated a nine-year-old boy with metastatic disease with reduced intensity trans- Figure 2 (a) CTL clones ( Â 10). (b) CTL clones ( Â 40). plantation. The patient developed a grade III GvHD and was alive showing no evidence of relapse 67 months after the hemopoietic SCT. Kounami et al.20 treated a 14-year- old boy with refractory osteosarcoma: the patient relapsed New experimental drugs on day þ 59 after transplantation and died of disease on Recent studies have been published on drugs not commonly day þ 197. used in ﬁrst-line treatments of osteosarcoma. Since no large series of studies are available, no Topotecan was employed as window therapy conclusions can be drawn on the effectiveness of non- in 26 metastatic osteosarcoma patients at diagnosis myeloablative SCT in osteosarcoma. followed by multi-agent chemotherapy, but it did not

Bone Marrow Transplantation Treatment of high-risk osteosarcoma F Fagioli et al S133 show an effective improvement as compared to the tumor cells, represents a potentially successful therapeutic traditional chemotherapy.21 approach to overcome the failure of patients to develop an Gemcitabine was employed alone with minimal re- efficient antitumor immune response.31 sponse,22 but in one study it was employed together with Recently, the expression of MAGE, SSX and SART-3 docetaxel in four patients with osteosarcoma, in which two family antigens has been identified in osteosarcoma cell partial responses have been observed.23 lines and in fresh osteosarcoma tissues, allowing the Ecteinascidin 743 has been employed alone in pretreated development of immunotherapeutic strategies in high-grade recurrent osteosarcoma patients with dismal results.24 osteosarcoma patients as well. Interferon as single-agent adjuvant therapy has been In this context, we intend to generate tumor antigen- used for 18months up to 5 years on 89osteosarcoma specific CTLs by stimulating patients’ PBMCs or CD8- patients in CR. The 10-year metastases-free survival was enriched populations with DCs loaded with irradiated 39% with a median follow-up of 12 years.25 The lack of a autologous apoptotic tumor cells or autologous tumor cell control group precludes conclusions. lysates (Figure 1). The novelty and relevance of this project consist in assessing the frequency by which antitumor T-cell lines can Conclusions be obtained ex vivo and in identifying which of the already known tumor antigens are recognized by these T-cell lines. These data indicate that only the combination of several The fate and function of tumor-reactive CTLs will be strategies, such as intensive chemotherapy to induce evaluated by analyzing the frequency of tumor-reactive remission followed by immunotherapy to maintain the CTL precursors (p) and IFN-g-secreting PBMCs by Elispot remission status, might improve the prognosis of these Assay, phenotype and cytokine release following CTL patients. generation (Figures 2a and b). On this basis, the AIEOP and the ISG devised a clinical trial for osteosarcoma patients with unresectable relapse consisting of three different phases: Summary algorithm

(a) Induction phase with chemotherapy and mobilization and PBSC apheresis to induce a CR or PR of the disease. OSTEOSARCOMA (b) High-dose samarium-153–EDTMO followed by PBSC reinfusion in patients with lesions positive at bone scintigraphy. In negative patients, the conditioning METASTA TIC OSTEOSARCOMA regimens consist of high-dose chemotherapy (carbopla- LOCALIZED OSTEOSARCOMA AT DIAGNOSIS AND RELAPSED tin and VP-16). OF THE EXTREMITIES METASTA TIC OSTEOSARCOMA (c) Immunotherapy phase in which patients with an HLA-identical sibling receive a non-myeloablative hemopoietic SCT (eventually followed by donor

lymphocyte infusion (DLI)) and patients without an TREATM ENT: NEW THERAPEUTIC OPTIONS HLA-identical sibling receive 12 cycles of low-dose IL-2. SURGE RY AND CHEMOTHERAPY UNDER STUDY

Future perspectives NEW HD EXPERIMENTAL CHEMOTHERAPY IMMUNOTHERAPY RADIOMETABOLIC DRUGS TREATMENT T-cell therapy has attracted a great deal of interest in the treatment of viral infections, EBV-associated tumors and TOPOTECAN NON-MYELOABLATIVE relapses of hematological disease. The persistence of SAMARIUM- GEMCITABINE allo-SCT 153-EDTMO adoptively transferred T cells and the reconstitution of ECTE INASCIDIN 743 IL-2 CTLs virus-specific immunity with no noticeable side effects have INTER FERON been amply demonstrated. Many solid tumors, such as sarcomas, express tumor- specific antigens on their surface, which can serve as targets for immune effector T cells. Nevertheless, the immune surveillance against clinically manifested tumors is rela- tively inefficient, and the number and the function of DCs Acknowledgements are dramatically reduced in patients with head and neck squamous cell carcinoma, breast, renal, colon and prostate This study has been partially supported by the Gigi Ghirotti cancer.26–28 The identification of tumor antigens and their Association and by Compagnia di S Paolo, Torino, Italy. recognition by tumor-specific lymphocytes have fueled the development of immunotherapeutic strategies in cancer.29,30 Conflict of interest Ex vivo generation/expansion, followed by in vivo infusion, of CTL lines or clones, able to selectively kill None of the authors declared any financial interests.

Bone Marrow Transplantation Treatment of high-risk osteosarcoma F Fagioli et al S134 References and non-malignant diseases. Transpl Immunol 2005; 14: 207–219. 1 Campanacci M. Bone Tumors, 2nd edn. Lippincott-Verlag: 16 Fagioli F, Ricchiardi A, Carnevale-Schianca F. Nonmyelo- Bologna, Italy, 1999, pp 1418–1468. ablative allogeneic hemopoietic stem cell transplants. Haema- 2 Bacci G, Ferrari S, Bertoni F, Ruggieri P, Picci P, Longhi A tologica 2002; 87: 13–19. et al. Long-term outcome for patients with nonmetastatic 17 Fagioli F, Berger M, Brach del Prever A, Lioji S, Aglietta M, osteosarcoma of the extremity treated at the Istituto Ferrari S et al. Regression of metastatic osteosarcoma Ortopedico Rizzoli according to the Istituto Ortopedico following non-myeloablative stem cell transplantation. A case Rizzoli/osteosarcoma-2 protocol: an updated report. J Clin report. Haematologica 2003; 88: ECR16. Oncol 2000; 18: 4016–4027. 18Carnevale-Schianca F, Cignetti A, Capaldi A, Vitaggio K, 3 Bielack S, Kempf-Bielack B, Schwenzer D, Birkfellner T, Vallario A, Ricchiardi A et al. Allogeneic nonmyeloablative Delling G, Ewerbeck V et al. Neoadjuvant therapy for hematopoietic cell transplantation in metastatic colon cancer: localized osteosarcoma of extremities. Results from the tumor-specific T cells directed to a tumor-associated antigen Cooperative Osteosarcoma Study Group COSS of 925 are generated in vivo during GVHD. Blood 2006; 107: patients. Klin Padiatr 1999; 211: 260–270. 3795–3803. 4 FerrariS,BriccoliA,MercuriM,BertoniF,PicciP,TienghiAet al. 19 Goi K, Sugita K, Tezuka T, Sato H, Uno K, Inukai T et al. A Postrelapse survival in osteosarcoma of the extremities: prognostic successful case of allogeneic bone marrow transplantation for factors for long-term survival. JClinOncol2003; 21: 710–715. osteosarcoma with multiple metastases of lung and bone. Bone 5 Voute PA, Souhami RL, Nooij M, Somers R, Cortes-Funes H, Marrow Transplant 2006; 37: 115–116. van der Eijken JW et al. A phase II study of cisplatin, 20 Kounami S, Nakayama K, Yoshiyama M, Yoshimasu T, ifosfamide and doxorubicin in operable primary, axial skeletal Aoyagi N, Yoshikawa N. Non-myeloablative allogenic and metastatic osteosarcoma. European Osteosarcoma peripheral blood stem cell transplantation in a patient Intergroup (EOI). Ann Oncol 1999; 10: 1211–1218. with refractory osteosarcoma. Pediatr Transplant 2005; 9: 6 Harris MB, Gieser P, Goorin AM, Ayala A, Shochat SJ, 342–345. Ferguson WS et al. Treatment of metastatic osteosarcoma at 21 Seibel N, Krailo M, Sato J. Phase II window of topotecan in diagnosis: a Pediatric Oncology Group Study. J Clin Oncol newly diagnosed metastatic osteosarcoma CCG 7943. Proceed- 1998; 16: 3641–3648. ings of the ASCO, May 12–15, 2001, San Francisco, CA, 7 Longhi A, Fabbri N, Donati D, Capanna R, Briccoli A, (abstract 1509). Biagini R et al. Neoadjuvant chemotherapy for patients with 22 Okuno S, Ryan LM, Edmonson JH, Priebat DA, Blum RH. synchronous multifocal osteosarcoma: results in eleven cases. Phase II trial of gemcitabine in patients with advanced J Chemother 2001; 13: 324–330. sarcomas (E1797): a trial of the Eastern Cooperative Oncology 8Bielack SS, Kempf-Bielack B, Delling G, Exner GU, Flege S, Group. Cancer 2003; 97: 1969–1973. Helmke K et al. Prognostic factors in high-grade osteosarcoma 23 Leu KM, Ostruszka LJ, Shewach D, Zalupski M, Sondak V, of the extremities or trunk: an analysis of 1702 patients treated Biermann JS et al. Laboratory and clinical evidence of on Neoadjuvant Cooperative Osteosarcoma Study Group synergistic cytotoxicity of sequential treatment with gemcita- protocols. J Clin Oncol 2002; 20: 776–790. bine followed by docetaxel in the treatment of sarcoma. J Clin 9 Bacci G, Ferrari S, Longhi A, Picci P, Mercuri M, Alvegard Oncol 2004; 22: 1706–1712. TA et al. High dose ifosfamide in combination with high dose 24 Laverdiere C, Kolb EA, Supko JG, Gorlick R, Meyers PA, methotrexate, adriamycin and cisplatin in the neoadjuvant Maki RG et al. Phase II study of ecteinascidin 743 in heavily treatment of extremity osteosarcoma: preliminary results of an pretreated patients with recurrent osteosarcoma. Cancer 2003; Italian Sarcoma Group/Scandinavian Sarcoma Group pilot 98: 832–840. study. J Chemother 2002; 14: 198–206. 25 Muller CR, Smeland S, Bauer HC, Saeter G, Strander H. 10 Fagioli F, Aglietta M, Tienghi A, Ferrari S, Brach del Prever Interferon-alpha as the only adjuvant treatment in high-grade A, Vassallo E et al. High-dose chemotherapy in the treatment osteosarcoma: long term results of the Karolinska Hospital of relapsed osteosarcoma: an Italian Sarcoma Group Study. series. Acta Oncol 2005; 44: 475–480. J Clin Oncol 2002; 20: 2150–2156. 26 Hoffman TK, Muller-Berghaus J, Ferris RJ, Johnson JT, 11 Sauerbrey A, Bielack S, Kempf-Bielack B, Zoubek A, Storkus WJ, Whiteside TL. Alterations in the frequency of Paulussen M, Zintl F. High-dose chemotherapy (HDC) and dendritic cell subsets in the peripheral circulation of patients autologous hematopoietic stem cell transplantation (ASCT) as with squamous cell carcinomas of the head and neck. Clin salvage therapy for relapsed osteosarcoma. Bone Marrow Cancer Res 2002; 8: 1787–1793. Transplant 2001; 27: 933–937. 27 Della Bella S, Gennaro M, Vaccari M, Ferraris C, Nicola S, 12 Franzius C, Bielack S, Sciuk J, Vollet B, Jurgens H, Schober O. Riva A et al. Altered maturation of peripheral blood dendritic High-activity samarium-153-EDTMP therapy in unresectable cells in patients with breast cancer. Br J Cancer 2003; 89: osteosarcoma. Nuklearmedizin 1999; 38: 337–340. 1463–1472. 13 Anderson PM, Wiseman GA, Dispenzieri A, Arndt CA, 28Gabrilovich D. Mechanisms and functional significance of Hartmann LC, Smithson WA et al. High-dose samarium-153 tumour-induced dendritic-cell defects. Nat Rev Immunol 2004; ethylene diamine tetramethylene phosphonate: low toxicity of 4: 941–952. skeletal irradiation in patients with osteosarcoma and bone 29 Bollard CM, Savoldo B, Rooney CM, Heslop HE. Adoptive metastases. J Clin Oncol 2002; 20: 189–196. T-cell therapy for EBV-associated post-transplant lymphopro- 14 Luksch R, Perotti D, Cefalo G, Gambacorti Passerini C, liferative disease. Acta Haematol 2003; 110: 139. Massimino M, Spreafico F et al. Immunomodulation in a 30 Pardoll D. T-cells take aim at cancer. Proc Natl Acad Sci USA treatment program including pre- and post-operative inter- 2002; 99: 15840. leukin-2 and chemotherapy for childhood osteosarcoma. 31 Montagna D, Schiavo R, Gibelli N, Pedrazzoli P, Tonelli R, Tumori 2003; 89: 263–268. Pagani S et al. Ex-vivo generation and expansion of anti-tumor 15 Resnick IB, Shapira MY, Slavin S. Nonmyeloablative cytotoxic T-cell lines derived from patients or their HLA- stem cell transplantation and cell therapy for malignant identical sibling. Int J Cancer 2004; 110: 76–86.

Bone Marrow Transplantation