Aglietta, Massimo

Berger, M

Brach Del Prever, A

Fagioli, F

Ferrari, S

Lioji, S

Madon, Enrico

Picci, P

English

Institutional Research Information System University of Turin

F. Fagioli et al.| 64 | haematologica/the hematology journal | 2003; 88(online)Regression of Metastatic Osteosarcoma FollowingNon-Myeloablative Stem Cell Transplantation.A Case ReportWe report the evidence of regression of multiplemetastases following non-myeloablative stem celltransplantation (NST) from an HLA-identical sib-ling in a case of relapsed fibroblastic osteosarco-ma. The course of NST was well tolerated. Fulldonor chimerism was achieved on day +150 bothfor CD15+ and CD3+ cells. Complete remissionwas achieved on day +116. On day +210 thepatient relapsed with a scapular metastasis thatwas unresponsive to four doses of donor lympho-cyte infusion (DLIs).To our knowledge, this is thefirst reported case showing the achievement ofcomplete remission following NST in an osteosar-coma patient.Haematologica 2007; 88:(5)e64-e66Introduction.Relapsed osteosarcoma with multiple bone metastasesis generally considered incurable using conventionaltreatment.1 High dose chemotherapy and autologousstem cell transplantation (ASCT) have improved theremission rate but convincing evidence of disease freesurvival (DFS)2 has never been shown. Progress in theunderstanding of the immune response to cancer togeth-er with an increased understanding of the basic mecha-nism of cellular immunology have combined to opennew opportunities for the development of effectiveimmunotherapies for cancer.Patients with leukaemiaundergoing allogeneic stem cell transplantation (SCT)and developing acute and, especially chronic GvHD runa lower risk of relapse than those without GvHD.3Furthermore, T cell depletion of the graft increases therisk of relapse. Recent reports suggest that donor lym-phocytes, transferred with the graft, may also produce aclinically meaningful graft versus tumour (GvT) effect inpatients with refractory solid tumours, such as renal cellcarcinoma,4 breast cancer,5 colonic cancer,6 non-small celllung cancer,7 ovarian cancer8 and Ewing sarcoma.9 Basedon these data, attempts have been made to diminishtransplant related mortality and, possibly, morbidityassociated with conventional myeloablative allogeneicstem cell transplantation. Recently, attention has beenaddressed to less cytotoxic conditioning regimens toobtain a mixed chimerism over which allogeneic cellsmight display an anti-tumour effect.In this report, wedescribe the evidence of the regression of multiplemetastases following non-myeloablative transplantation(NST) in a case of relapsed fibroblastic osteosarcoma.Patient and Donor.In February 1997 a 12 year-old boy was diagnosed agrade IV fibroblastic osteosarcoma of the right distalfemur. No metastasis was found at diagnosis. Hereceived neo-adjuvant chemotherapy according to theISG-SSG1 protocol (an Italian-Scandinavian chemothera-py and surgery protocol) including high doseMethotrexate, high dose Ifosfamide, Adriamycin andCis-platinum. In June 1997 he underwent resection ofthe distal femur and insertion of a Kotz prothesis. Thehistological examination showed 100% of tumournecrosis. The patient finished post-surgery chemothera-py in October 1997. The Adriamycin, Methotrexate,Cisplatinum and Ifosfamide total doses administratedwere 330 mg/m2, 48 g/m2, 480 mg/m2 and 60 g/m2,respectively. In October 2000 the patient underwent leftlung metastasectomy (+ 44 months from diagnosis). InJune 2001 (+ 52 months from diagnosis) the total bodyscan with 99Tc showed multiple osseous metastases. ACT scan showed a retro-orbital mass, whereas the lungswere negative (Tab 1).10 Neoplastic cells were also foundin the bone marrow biopsy.The patient underwent amodified ISG-AIEOP (Italian Sarcoma Group-ItalianAssociation of Paediatric Haematology Oncology) forvery high risk osteosarcoma patients, that was approvedby the Hospital Ethical Committee. The patient wastreated with three cycles of high dose Methotrexatecycles (12 g/m2), a Cisplatinum-Adriamycin cycle (120mg/m2 and 75 mg/m2, respectively) and a Cis-platinumcycle (120 mg/m2). In November 2001 the total bodyscan with 99Tc-Osteosol showed the partial uptakereduction, while the retro-orbital cavity CT scan wasunchanged. As he had several healthy siblings, his 22-year-old HLA-identical brother was chosen as a stem celldonor.Allogeneic stem cell transplantation. The patient receivedFludarabine 30 mg/m2/day over four days andCyclophosphamyde 30 mg/Kg/day over two days. Thegraft versus host disease (GvHD) prophylaxis was:Cyclosporin-A (Cy-A) 3 mg/Kg i.v. from day -1 to +84 tomaintain blood values between 100-250 ng/ml andMycofenolate Mofetil (MMF) 15 mg/Kg b.d. orally fromday 0 to +28. Cy-A tapering was started from day +84until day +180 (7% weekly). Acyclovir was given forviral prophylaxis 500 mg/m2 three times a day from day&endash;5 and liposomal anphotericin 1 mg/Kg wasgiven for antifungal prophylaxis from day -1. ForPneumocystis carinii prophylaxis aerosolized pentami-dine 300 mg and, thereafter, trimethoprim-sulfametoxa-zole 5 mg/Kg over two consecutive days each week wasgiven.The graft was: nucleated cells 10x108/Kg, CD34+13.8x106/Kg, CFU-GM 60x104/Kg, CD3+ 2.22x108/Kg,CD3+CD4+ 1.04x108/Kg, CD3+CD8+ 1.02x108/Kg,CD19+ 2.8x107/Kg, CD56+ 3.2x107/Kg.Immune reconstitution following. NST. Monthly, peripher-al blood samples were analysed for CD3+, CD4+, CD8+,CD19+ and CD56+ lymphocyte reconstitution. Cellswere analysed on a Becton Dickinson Facscan cytometer.Molecular studies to monitor chimerism. DNA from patientTable 1.and donor pre-transplant samples was obtained using astandard protocol. (Qiagen, Hilden, Germany). The sep-aration of CD3+ and CD15+ cells from peripheral bloodwas performed with immunomagnetic beads (MiltenyiBiotec srl, Italy). Before freezing, 30 mM Tris buffer wasadded to each cell pellet. After thawing, 60 µL of lysisbuffer was added to each bead-selected cell sample.These samples were then digested at 37°C overnight ona shaker, and subsequently diluted, as previouslydescribed elsewhere. The cell lysate samples were heat-ed to 70°C, then spun and cooled on ice. Polymerasechain reaction (PCR) amplification of two different vari-able number tandem repeats (VNTRs) were chosen formixed chimerism analysis.Evaluation of tumour regression. Table 1 shows theresults of the re-staging performed periodically aftertransplantation.Results.Transplant related toxicity. The patient did not developacute and chronic GvHD. Transplant related toxicity was0 according to the Bearman score.Haemopoietic engraftment. The patient achieved neu-trophil engraftment (>500/µL) on day +14. The lowerplatelet count was 119.000/µL. One packed red celltransfusion was performed on day +1 (before transfusionHgb level 8.1 g/dL).Immune reconstitution following NST. The CD4+ cellreconstitution was monitored from day +27, howeversince day+16 the total lymphocytes were >500/µL. Asreported in Table 2, also the CD19+ population showeda quick recovery following NST.Chimerism analysis. The PCR study for the VNTRsshowed from day+18 >50% of recipient cells in the bonemarrow. On day +37 the peripheral blood analysis formixed chimerism showed CD15+ and CD3+ cells with>60% of donor origin and on day +84 the unseparatedbone marrow cells, the peripheral blood CD15+ andCD3+ cells showed >70% of donor origin. On day +150the patient achieved full donor chimerism. No evidenceof rejection emerged, even at the time of relapse.Tumour response. On day +116 the head CT scanshowed complete remission of the retro-orbital mass,and the total body scan with 99Tc-Osteosol showed noactive metastases. The complete remission status wasmaintained until day +180. On day +210 following NST,the patient relapsed with a single right scapular metasta-sis. He received four, monthly, escalating doses of donorlymphocyte infusion (DLIs): 1x107/Kg, 5x107/Kg,7x107/Kg, 1x108/Kg CD3+ lymphocytes. No tumourresponse or GvHD developed. The patient is now alivewith progressive disease on day +420 following trans-plantation.Discussion.Patients with osteosarcoma and multiple bone metas-tases have been shown to have a DFS of 0% at 4 yearsfollow-up 1. In a recently reported series, for patientswith early relapse, high dose chemotherapy with stemcell rescue and pre or post-surgery, had a high remissionrate, but DFS remained low (12%) at 3 year follow-up 2.Here, we report the evidence of metastases regression ina patient with advanced osteosarcoma, in which theimmunological anti-osteosarcoma effect was independ-ent of the GvHD reaction. Evidence that regression ofmetastatic osteosarcoma cells was mediated by the graftversus tumour (GvT) effect is compelling. First, theFludarabine and Cyclophosphamide preparative regimenwas administrated at immunosoppressive dosages, toallow the donor’s immune cell engraftment, while avoid-ing the substantial side-effects of myeloablative therapy.Indeed, the conditioning regimen did not affect thetumour growth as shown by the re-staging done on day+30. Secondly, the regression of metastatic sites wasdelayed as complete remission was not achieved beforeday +116 following NST. Thirdly, the anti-tumourresponse developed only after a mixed chimerism statuswas obtained. A greater immunological differencebetween donor lymphocytes and recipient tumour cellsmay be the reason why allogeneic cells have an unequiv-ocal anti-osteosarcoma effect in the NST setting in theabsence of any signs of GvHD. This phenomenon mightbe explained considering that initially a mixed chimera isformed, and the recipient and host T and B lymphocyteand dendritic cells locate the thymus and delete host anddonor reactive T cells. This may result in a tolerant T cellrepertoire for both host and donor cells. Thus, theformed mixed-chimera serves as a platform for adoptiveimmunotherapy in which the risk for GvHD is reducedas the severity of its clinical manifestation is also relatedto the conditioning regimen and subsequent cytokinerelease. The GvT effect is maintained by the host antigenhaematologica online 2003haematologica/the hematology journal | 2003; 88(online) | 65 |Table 2.presenting cell, which elicits the overall GvT effect byrecognising neoplastic antigens to donor lymphocytes.We conclude that this first evidence of regression follow-ing NST suggests a role for immunological control ofosteosarcoma by allogeneic cells. Franca Fagioli, Massimo Berger, Adalberto Brach del Prever,Stefano Lioji, Massimo Aglietta, Stefano Ferrari , Piero Picci , EnricoMadonCorrespondence: Franca FagioliDepartment of Pediatrics, Piazza Polonia 94, 10126, Turin, Italy.Tel +39-011-3135230 Fax +39-011-3135487References1. Ward WG, Mikaelian K, Dorey F, Mirra JM, Sassoon A,Holmes EC, Eilber FR, Eckardt JJ.. Pulmonary metastasis ofstage IIB extremity osteosarcoma and subsequent pulmonarymetastases. J Clin Oncol 1994; 12: 1849-58.2. Fagioli F, Aglietta M, Tienghi A, Ferrari S, Brach del Prever A,Vassallo E, Palmero A, Biasin E, Bacci G, Picci P, Madon E.High dose chemotherapy in treatment of relapsed osteosarco-ma: an italian sarcoma study group. J Clin Oncol 2002; 15:2150-6.3. Locatelli F, Zecca M, Rondelli R, Bonetti F, Dini G, Prete A,Messina C, Uderzo C, Ripaldi M, Porta F, Giorgiani G, GiraldiE, Pession A. Graft versus host disease prophylaxis with low-dose cyclosporine-A reduces the risk of relapse in childrenwith acute leukemia given an HLA-identical sibling bone mar-row transplantation: results of a randomized trial. Blood 200095(5): 1572-9.4. Childs R, Chernoff A, Contentin N, Bahceci E, Schrump D,Leitman S, Read EJ, Tisdale J, Dunbar C, Linehan WM, YoungNS, Barrett AJ. Regression of metastatic renal-cell carcinomaafter nonmyeloablative allogeneic peripheral blood stem celltransplantation. N Eng J Med 2000; 343: 750-8.5. Eibl B, Schwaighofer H, Nachbaur D, Marth C, Gachter A,Knapp R, Bock G, Gassner C, Schiller L, Petersen F,Niederwieser D. Evidence for a graft versus tumor effect in apatient treated with marrow ablative chemotherapy and allo-geneic bone marrow transplantation for breast cancer. Blood1996; 88:1501-8.6. Zetterquist H, Hentschke P, Thorne A, Wernerson A, MattssonJ, Uzunel M, Martola J, Albiin N, Aschan J, PapadogiannakisN, Ringden O.. A graft-versus-colonic cancer effect of allo-geneic stem cell transplantation. Bone Marrow Transplant2001; 28:1161-6.7. Moscardo F, Martinez JA, Sanz GF, Jimenez C, Cervera J,Sanchis J, Vera FJ, Sanz MA.. Graft versus tumour effect innon-small cell lung cancer after allogeneic peripheral bloodstem cell transplantation. Br J Haematol 2000; 111: 708-710.8. Bay JO, Choufi B, Pomel C. Potential allogeneic graft versustumour in a patient with ovarian cancer. Bone MarrowTransplant 2000; 25: 681-682.9. Koscielniak E, Klingebiel T, Gross-Wieltsch U et al.Transplantation of a megadose of full-haplotype mismatchedhematopoietic stem cells (HSCT) as a treatment of metastaticrelapse of Ewing sarcoma-Report from the Committee onHigh Risk Sarcoma of the Cooperative Soft Tissue SarcomaStudy (CWS). Bone Marrow Transplant (abstract) 2002; 29(2):S191-2.10. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS,Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT,Christian MC, Gwyther SG.. New guidelines to evaluate theresponse to treatment in solid tumors. European Organisationfor Research and Treatment of Cancer, National CancerInstitute of the United States, National Cancer Institute ofCanada. J Natl Cancer Inst 2000; 92(3): 205-16.F. Fagioli et al.| 66 | haematologica/the hematology journal | 2003; 88(online)

Regression of metastatic osteosarcoma following non-myeloablative stem cell transplantation. A case report.

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Regression of metastatic osteosarcoma following non-myeloablative stem cell transplantation. A case report.

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