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Thiotepa, Busulfan, and Cyclophosphamide: A New Preparative Regimen for Autologous Marrow or Blood Stem Cell Transplantation in High-Risk Multiple Myeloma By Meletios A. Dimopoulos, Raymond Alexanian, Donna Przepiorka, Jeane Hester, Borje Andersson, Sergio Giralt, Rakesh Mehra, Koen van Besien, Kay B. Delasalle, Christopher Reading, Albert B. Deisseroth, and Richard E. Champlin Forty patients with multiple myeloma received thiotepa (750mg/m2),busulfan(10 mg/kg), and cyclophosphamide (12 0 mg/kg) (TBC)followed by autologous bone marrowor blood stem cell support. Granulocyte-Colony stimulating factor (G-CSF)was administeredto accelerate hematopoietic recovery. Sixty-five percent of all patients responded to this treatment. Eighty-eight percent of patients transplanted in partial remission had a further reduction of the myeloma and 53% achieved a complete remission. Fortyeight percent of patients with refractory myeloma responded. All responding patients transplanted during par- tial remission or with primary refractory myeloma remain free of progression for a period of 4 to 2 4 months posttransplant, but the remission duration of patients treated in refractory relapse was short (4 months). Five of 24 patients transplanted with marrow and none of 16 receiving blood stem cells died of treatment-related complications. Use of blood stem cells resulted in more rapid granulocyte and platelet recovery. W e conclude that TBC is an effective, relatively well tolerated, preparative regimen for patients with multiple myeloma. 0 1993 by TheAmerican Society of Hematology. DURING THE LAST decade, dose intensive therapy with autologous bone marrow or blood stem cell transplantation has emerged as a useful treatment for selected patients with multiple myeloma.'-4High-dose melphalan combined with total body irradiation (TBI)has been the most frequently used conditioning regimen. A marked cytoreduction has been achieved in the majority of patients and the outcome of those treated in early phases of their disease appears better than that of patients treated during refractory relapse.' Although radiotherapy is effective for multiple myeloma, higher doses are required for local control than can be administered as TBI. In addition, many patients are ineligiblefor TBI because of previous radiotherapy to the spine. Alkylating agents are active drugs for treatment of multiple myeloma. There has been considerable interest in combinations of alkylating agents with nonoverlapping extramedullary toxicities as preparative regimens for marrow transplantati~n.T~?h~e busulfan-cyclophosphamide combination has shown activity in patients with myeloma undergoing allogeneic bone marrow transplant,'^^ but this regimen has substantial toxicity, particularly hepatic venoocclusive disease in heavily pretreated patients."," Thiotepa is an active agent against multiple mye10ma;~it is Iimited by cutaneous and mucosal toxicity and at very high doses can have central nervous system effects.I2To determine if addition of thiotepa to a reduced dose of busulfan and cyclophosphamide would be a tolerable and effective preparative regimen, we conducted a phase I1 study of a From the Department of Hematology, the University of Texas M.D. Anderson Cancer Center, Houston, TX. Submitted February 8, 1993; accepted June 24, 1993. Address reprint requests to Richard E. Champlin, MD, Depart- ment of Hematology, Section of Bone Marrow Transplantation. 1515 Holcombe Blvd, Box 65, Houston, TX 77030. Thepublication costs of this article were defrayed in part bypage charge payment. This article must therefore be hereby marked "advertisement"in accordancewith 18 U.S.C.section 1734 solely to indicate thisfact. 0 1993 by The American Society of Hematology. 0006-4971/93/8208-002 7$3.00/0 combination of these three alkylating agents, with autologous bone marrow or stem cell transplantation. PATIENTS AND METHODS Eligibility criteria. Between May I99 1 and December 1992,40 consecutive patients with multiple myeloma were treated. The study was reviewed and approved by the University of Texas M.D. Anderson Institutional Review Board and all patients provided written informed consent. Eligibility criteria included patient age less than 65 years, performance status 1 2 on the Zubrod scale, and adequate organ function (creatinineclearance 250 mL/min, diffusion lung capacity 250% of predicted, and left ventricular ejection fraction 250%).Patients were treated during resistant relapse, primary resistance, or, in high-risk patients, during remission either after primary treatment or after salvage chemotherapy treatment. Autologousbone marrow was used when marrow plasma cells were less than 30%.The harvested marrow was required to have at least 1.5 x 108/kg normal nucleated marrow cells and 1 X IO4 colony forming unit-granulocyte macrophage (CFU-Gm)/kg. When less than 5% bone marrow plasma cells were present, the marrow was depleted of B-lymphoid precursors using anti-CDI9 monoclonal antibody and immunomagneticbeads." Patients with greater than 30%bone marrow plasmacytosis,prior radiotherapyto the pelvis or lumbosacral spine, or inadequate colony formingunits on marrow harvest were eligible for collection of blood progenitors by leukapheresis. Putients. Patient characteristics are shown in Table 1. Median age ofall patientswas 49 years (range, 34 to 64 years). Twenty-eight were male and 12 were female. Patients were staged as high, intermediate, and low tumor mass myelomaaccordingto standard criteria.I4Thirteen newly diagnosed patients who presented with either high or intermediate tumor mass were transplanted in first partial remission. Four patients who were resistantto melphalan and prednisone responded to a subsequent vincristine, doxorubicin, dexamethasone (VAD)-rescue treatment and were transplanted in second partial remi~sion.'N~o patient was in complete remission before myeloablative treatment. Fifteen patients, classified as primary refractory, had never responded to standard therapy and VAD; 10 of them were also resistant to a high-dose cyclophosphamide-etoposidesalvage regimen.I6Eight patients were transplanted during relapsedespite a salvagetreatment (refractoryrelapse).Nine of the 40 patients had previouslyreceived radiotherapyto the spine at doses precluding use of a TBI-containingablative regimen. Autologous marrow. Eleven patientstransplanted in first or second partial remission with less than 5% marrow plasma cells received autologousbone marrow depleted of B-lymphoidprecursors 2324 Blood, Vol82, No 8 (October 15). 1993: pp 2324-2328 THIOTEPA, BUSULFAN, AND CYCLOPHOSPHAMIDE Table 1. Patient Characteristics at Transplantation First Partial Remission Second Partial Remission No. Median age (range) Tumor mass High or intermediate &-microglobulin >3 mg/dL Median months from initial treatment (range) Source of stem cells Unpurged marrow CDl9-purged marrow Blood stem cells 13 51 (34-64) 0 3 5 (2-12) 0 9 4 4 44 (37-49) 0 0 2 0 (13-28) 0 2 2 Primary Refractory 15 46 (37-60) 9 8 9 (3-25) 5 0 10 2325 Refractow Relapse 8 5 2 (38-59) 4 6 21 (17-52) 8 0 0 using anti-CD19 monoclonal antibody and immunomagnetic beads.13Percoll separated mononuclear cells were incubated with IgG anti-CD19 monoclonalantibody at 4C for 30 minutes at I X 10' cells/mL. Sheep antimouse IgGl conjugated magnetic beads (Dyna-beadsM-450) were added at a concentration of 2 beads per cell and incubatedfor 30 minutes at 4C on a tiltingdevice.The cell suspension was passed through the separation device at a flow rate of 20 mL/min at 4C into a collection bag and then cryopreserved in 10% DMSO using standard techniques. The percentage of CD19+cells in the percoll separated fractionsranged from 0.2% to 14.3%(median, 2.9%) before immunomagnetic purging. After this procedure, 6 of 11 patients did not have demonstrable CD19+by immunofluorescence while the remaining patients had between 0.2% and 0.8% residual CD19 cells. Immunomagnetic purging resulted in a median recovery of 80.7%CD34' cells(range, 36.9%to 100%)after the procedure. Five patients with primary refractory myeloma were transplanted with unmanipulated autologousbone marrow, which contained less than 30% plasma cells, whereas IO patients with more heavily infiltrated marrow received autologousblood stem cells. All patients with myeloma in refractory relapse received unmanipulated autologous bone marrow that was harvested during a prior remission when bone marrow plasma cells were less than 5%. Blood stem cell collection. Six patients in first or second partial remission who had previouslyreceived radiotherapyto the pelvis or lumbosacral spine and 10 patients with marrow plasmacytosis greater than 30%underwentblood stem cell collection. All patients were hospitalized. A double lumen quinton catheter was inserted into a subclavian vein and a combination of cyclophosphamide3.0 g/mZintravenously (IV) and etoposide900 mg/m2IV was administered over 5 days as previouslydescribed.16On day 6, GM-CSFwas started at a dose of IO pg/kg subcutaneously daily. Apheresis of blood mononuclearcellswasperformedwith a Cobe Spectrablood cell separatorand commencedduringhematopoietic recovery when the absolute mononuclear cell (mnc) count was greater than 400/pL. Stem cell collection was performed daily to obtain at least 4 X 10' mnc/kg body weight. Five patients required platelet transfusionsto facilitate stem cell collection. Conditioning regimen and supportive care. The ablative regimen consisted of thiotepa 250 mg/m2 in 250 mL normal saline (NS) IV over 4 hours on days -9, -8, and -7; busulfan 1.0 mg/kg orally (PO) every 6 hours for IO doses on days -6, -5, and -4; cyclophosphamide 60 mg/kg in 200 mL dextrose 5% in water (DSW)IV over 1hour through a centralvenouscatheteron days -3 and -2. Mesna 250 mg/m2 was administered 30 minutes before and every 4 hours for six doses after each cyclophosphamideinfusion. Autologousmarrow or blood stem cellswere reinfused on day 0 through a central venous catheter preceded by methylpredniso- lone 100 mg IV and benadryl 50 mg IV. On day + I , G-CSF was started at a dose of IO pg/kg/d, which was continued until granulocyte count exceeded 4,OOO/pLfor 3 consecutive days. The doses of thiotepa, busulfan, and cyclophosphamide (TBC) were those defined in a phase I study in patients with advanced hematologic malignancies." All patients were treated in a private room. Antibiotic prophylaxis included daily oral norfloxacin 800 mg and IV vancomycin 1 g/d in addition, acyclovir 15 mg/kg and fluconazole 400 mg were administered daily IV. Patients received IV Ig at a dose of 500 mg/kg every 2 weeks starting on day -8. Toxicity grading. All cases of nonhematologic organ dysfunction were considered regimen related toxicities (RRT) unless they could be clearlyexplained by another cause (eg, renal failuredue to septic shock, respiratory failure due to documented pneumonia). The grading scale described by Bearman et al was used." Grade 0 represented no toxicity; grade I toxicity was fully reversiblewithout specific intervention; grade 2 toxicity was not life-threatening, but its reversal required specific measures; grade 3 toxicity was lifethreatening but reversible;and grade 4 toxicity was fatal. Response. Serial measurements of blood counts, chemistries, and electrophoretic studies were conducted. Complete remission (CR)was definedasdisappearanceof serum monoclonal protein on immunofixationand no evidence of monoclonalplasma cellsin the bone marrow for at least 2 months. Partial remission (PR) required 275% reduction of serum myeloma protein synthesis with disappearanceof Bence Jones proteinuria and reduction of bone marrow plasmacytosisto less than 5%for at least 2 months.19Patients who died of transplant-relatedcomplications(toxicdeaths)were considered treatment failures. Maintenance therapy. Alpha-interferon at a dose of I X IO6 U subcutaneously three times a week was started when the granulocyte count recovered to greater than 2,5OO/pL and platelets r50,000/pL. The dose of interferon was increased as tolerated to 2 X IO6 U/m2 three times a week. In addition, dexamethasone 20 mg/m2 daily was administered PO for 4 days each month. This maintenance program was continued until there was evidence of disease progression. RESULTS Hematologic recovery. All patients experienced profound pancytopenia. Hematologic recovery is summarized in Table 2. G-CSFwas well tolerated and its administration was not discontinued in any patient. Mediantime to granulocyte and platelet recovery was similar in patients transplanted with unmanipulatedor CD19-purged marrow. One patienttransplanted in second remissionwith CD19-purged 2326 DIMOPOULOS ET AL Table 2. Hematologic Recovery Posttransplant Median Davs Iranae) Platelets Stem Cell Source Granulocytes No. >500/rL >25.000/rL >50,0OO/rL Unpurged marrow ~~~ 13 15 (10-29) 35 (1 1-49) 41 (17-56) CD19-purged marrow 1 1 13 (10-36) 27 (16-39) 32 (16-69) Blood stem cells 16 9 (7-14) 12 (8-58) 15 (9-59) marrow did not show signsof granulocyteengraftment and bone marrow biopsy showed aplasia on day 21 posttransplant; this patient subsequently recovered when her unpurged marrow was infused. The time to recovery of granulocytes greater than 500/pL and platelets greater than 25,OOO/pL was compared among patients transplanted with bone marrow (purged or unmanipulated) with that of patients receiving stem cells (Table 2). Recovery of granulocytes and platelets was significantly more rapid with blood stem cell transplantation ( P = .OOO I ). Toxicity. Thirty-five of the 40 patients developed at least grade 1 toxicity (Table 3). The most frequent toxicities were stomatitis and diarrhea, but none developed higher than grade 3 toxicity of the gastrointestinaltract. Four patients (10%)developed grade 3 or 4 RRT, which was fatal in two patients. Eight patients developed thiotepa skin toxicity, which was characterizedby pruritus and dermatitis predominantly involving the axillary folds. The skin toxicity was associated with exfoliation in three patients. Pigmentation and tender swelling of the palmar and plantar surfaces occurred in two patients. Cutaneous toxicity was reversible in all patients. All but three patients developed neutropenic fever. Blood cultures showed gram-positive organisms in seven patients and gram-negative organisms in five; three patients developed documented sinopulmonary aspergillosis. Three patients developed cytomegalovirus (CMV) infection including 1 patient with fatal CMV pneumonia. Five patients ( 13%) experienced a treatment-related death, All had been transplanted with autologous marrow. Their median age was 52 years (range, 41 to 59 years). Two had resistant relapse myeloma, two had primary refractory disease, and 1 patient was transplanted in first remission. Two patients died from pneumonia developing during the aplastic phase; 1 patient died of hepatic venoocclusivedisease, and another patient of diffuse alveolar hemorrhage. The fifth patient expired from CMV pneumonia on day 67 posttransplant. None of the patients transplanted with blood stem cells expired. Responseand survival. Among 13 patients transplanted in first PR, 11 had further reduction ofthe myeloma protein and seven patients achieved a CR (Table 4). All four patients who were transplanted during a second PR showed marked reduction of the myeloma protein with a CR in two patients. Except for one therapy-related death, all patients transplanted in first or second PR remain free of progression from 4 to 20 months posttransplant (Fig 1). Seven of 15 patients with primary refractory myeloma responded; one achieved CR and 6 a PR. Six failed to respond and two died of transplant-related complications. The median time to achieve remission was 0.9 month (range, 0.1 to 5.0 months) posttransplant. Ten primary refractory patients were not only resistant to standard alkylating agents and VAD, but also failed to respond to cyclophosphamide (3 g/m2) and etoposide (900 mg/m2). Five such patients responded to the TBC regimen. All responding patients remain free of progression (Fig I). Four of eight patients with refractory relapse myeloma achieved a PR. Two failed to respond and two died of treatment-related complications.Responsesoccurred after a median of 0.3 month (range, 0.1 to 0.7). The remission duration in this categorywas short (median, 4. l months) and the median survival time after transplant was only 4 months. Alpha interferon was started at a median day 41 (range, 13 to 76 days) posttransplant in responding patients and was well tolerated with the exception of three patients in whom reversible leukopenia required temporary discontinuation of this therapy. The interferon dose was escalated to 2 X IO6 U/m2three times a week in all patients. DISCUSSION The combination of thiotepa, busulfan, and cyclophosphamide was designed with a goal to optimize dose intensity by combining three active alkylating agents with nonoverlappingtoxicity for treatment of high-risk patients with multiple myeloma. In addition, an opportunity was developed for future studies of repeat transplant-supported therapies in selected patients. The TBC regimen allowed us to treat nine patients who had previously received radiotherapy to the spine in doses that precluded TBI. Table 3. Regimen-Related Toxicity Grade 012 Mucositis 8 21 1 1 Gastrointestinal 24 14 2 Liver 28 7 1 Cardiac 37 1 2 CNS 37 0 0 Renal 37 1 0 Bladder 37 1 1 Pulmonary 36 1 1 3 Table 4. Clinical Outcome After Transdant Myeloma 4 Early Protein >75% No No. Death Reduction (CR) Response First partial remission Second partial remission Primary refractory Refractory relapse Total 13 4 15 8 40 1 0 2 2 5 1 1 (7) 4 (2) 7 (1) 4 (0) 26 (10) 1 0 6 2 9 THIOTEPA, BUSULFAN, AND CYCLOPHOSPHAMIDE 2327 100 Remission (17) I ,I II 1 ,I , I, .c0- 50 .-v) v) E 2 .C- c, C :0) a 20 -7 L Primary Refractory (15) 10 + : : : : ! : : : : : : : : : : : : : : : : : : I 5 10 15 20 Months after Transplant 25 Fig. 1. Progression-free survival for different groups of patients with multiple myeloma; remission curve includes patients in first and second remission. Failure includes no response, relapse, or death from any cause. Numbers in parentheses refer to patients in each category. Each of the drugs used in this combination has proven activity against myeloma and a different spectrum of extra- medullary toxicities. The combination of busulfan and cy- clophosphamide with allogeneic marrow transplantation is widely used for patients with leukemia,20as well as in some patients with multiple High-dosebusulfan with autologousbone marrow rescue has been recently reported to have activity against resistant myeloma." To decrease the relatively high incidence of regimen-related toxicity,we reduced the dose of busulfan and introduced thiotepa, which when administered at high doses, is active against mye10ma.I~Furthermore, there is in vitro and in vivo evi- dence of synergism between cyclophosphamide and thio- tepa.22 The TBC combination was relatively well tolerated. Re- versible skin toxicity, presumably due to thiotepa, occurred in eight patients. Three patients developed higher than grade 2 liver toxicity. No patient developed regimen-related central nervous system side effects. Thirteen percent of pa- tients died as a complication of the treatment, a frequency similar to that observed with melphalan/TBI or other non- TBI-containing regimens!-6 This trialkylator combination induced a marked cytore- duction of the myeloma in approximatelytwo-thirds of our patients. Using strict criteria, which include negative serum immunofixation, 53% of patients transplanted during first or second PR achieved a CR and myeloma has not yet re- lapsed in any patient. The TBC regimen induced PR or CR in 50%of patients resistant not only to standard treatments, but also to a high-dosecyclophosphamide-etoposidesalvage therapy.16The efficacy of myeloablativetherapy in patients with primary refractorymyeloma, especiallywhen adminis- tered during the first year of treatment, has been previously r e p ~ r t e dW. ~hile one-half of patients transplanted during a refractory relapse phase of myeloma responded, remission duration and subsequent survival were short. This is similar to the experience with melphalan and TBI in this category of patients; such patients have not had meaningful benefit from high-dose therapies and alternative approaches are needed.5 The optimal source of autologous stem cell for transplan- tation in patients with multiple myeloma has not been clearly defined and both marrow and blood stem cells were used in a recent Peripheral blood progenitors col- lected by multiple leukapheresis were the only source of cells for 16 patients with heavily infiltrated bone marrow or with prior radiotherapy to the pelvis or lumbosacral spine. Recovery of normal hematopoiesis occurred earlier than with bone marrow transplantation, and none died from treatment complications, suggesting that blood stem cells may be preferable for the support of ablative therapy for myeloma patients, assuming remission duration is not ad- versely affected. Eleven patients were transplanted in partial remission us- ing autologous marrow depleted of B-lineage cells using an anti-CD 19monoclonal antibody and immunomagnetic sep- aration. All but one patient engrafted and the timing of hematopoietic reconstitution was similar to that of patients receiving unmanipulated marrow. Sixty-four percent of such patients achieved CR. A similar study reported re- cently by Anderson et a124also demonstrated the feasibility of this approach. Controlled trials are necessary to define the role of autologous marrow purging in this disease. Whether purging of blood stem cells is feasibleor necessary also remains to be determined. Interferon-a and dexamethasone are both active agents against myeloma especially when administered during re- mission or to patients with low tumor mass d i ~ e a s e . ' ~A,l~- ~ though high-dose therapy with autologous transplantation is effective to produce CR, this approach has not been cura- tive. Posttransplant therapy with these agents was included to suppressresidual myeloma likely to be present after high- dose therapy. The impact of maintenance therapy after au- tologoustransplantation can only be assessed in the context of a controlled trial. A preliminary analysisof a randomized trial in myeloma reported that interferon prolongedthe pro- gression-free survival when administered after autologous bone marrow tran~plantation.~' In conclusion, this study indicates that this triple alkyla- tor combination regimen is effective to produce extended remissions in selected patients with multiple myeloma. Controlled studiesinvolvinglarger numbers of patients with extended follow-up are required to define its role and the optimal timing of the procedure. A high degree of cytore- duction occurred in patients treated during PR or with pri- mary refractory myeloma. Patients with myeloma in refrac- 2328 DIMOPOULOS ET AL tory relapse had only transient responses and do not appear to benefit from currently available ablative therapies; alternative approaches should be studied in these patients. ACKNOWLEDGMENT The authors are grateful to Eva Barcelb for excellent secretarial assistance. REFERENCES I. Barlogie B, Alexanian R, Dicke K, Zagars G, Spitzer G, Jagannath S, Honvitz L: High dose chemoradiotherapy and autologous bone marrow transplantation for resistant multiple myeloma. Blood 702369, 1987 2. Fermand JP, Levy Y, Gerota J, Benbunan M, Cosset TM, Castaigne S,Seligman M, Brouet J C Treatment ofaggressive multiple myeloma by high-dose chemotherapy and total body irradiation followed by blood stem cell autologous graft. Blood 73:20, 1989 3. Attal M, Huguet F, Schlaifer D, Payen C, Laroche M, Fournie B, Mazieres B, Pris I, Laurent G: Intensive combined therapy for previously untreated aggressive myeloma. Blood 79: I 130, 1992 4. 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Mandelli F, Avrisati G, Amadori S, Boccadoro M, Gernone A, Lanta V, Marmont F, Petrucci M, Tribalto M, Vegna M, Dammaco F, Pileri A: Maintenance treatment with recombinant interferon alfa-2b in patients with multiple myeloma responding to conventional induction chemotherapy. N Engl J Med 322:1430, 1990 26. Alexanian R, Dimopoulos MA, Delasalle K, Barlogie B: Primary dexamethasone treatment of multiple myeloma. Blood 80:887, 1992 27. Cunningham D, Powles R, Malpas JS, Milan S, Meldrum M, Viner C, Montes A, Hickish T, Nicolson M, Johnson P, Mansi J, Treleavan J, Raymond J, Gore ME: A randomized trial of maintenance therapy with Intron-A following high dose melphalan and ABMT in myeloma. Proc Am SOCClin Oncol 12:364, 1993