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High-Dose Melphalan Transplantation With Autologous Bone Marrow for Multiple Myeloma By Bart Barlogie, Roy Hall, Axel Zander, Karel Dicke, and Raymond Alexanian A large dose of melphalan was given to 23 patients with advanced multiple myeloma that was refractory to multiple prior treatments. Sixteen patients received a dose of 80 to 100 mg/m2, and seven were given 140 mg/rn2 followed by autologous bone marrow infusion. Tumor mass was reduced by more than 75% in 1 4 patients. including four who died of bone marrow aplasia. Serious infections were prevented in six of seven patients who received autologous bone marrow. The marked cytoreduction in patients with U NTIL RECENTLY, few treatment programs have been effective in patients with multiple myeloma resistant to alkylating agent-prednisone combinations. Using frequent courses of glucocorticoids with vincristine and doxorubicin by continuous infusion (VAD), about one half of our patients achieved a marked tumor reduction.' These results indicated that both dose and schedule modifi- cation of standard drugs would enhance tumor cell kill, a finding that was supported by the marked cytoreduction achieved in most patients with very high doses of melphalan.2 To improve the results from salvage therapies, we evaluated high doses of melphalan in 23 patients with advanced multiple myeloma that was resistant to both standard mel- phalan and the VAD regimen. Sixty percent of our patients showed rapid and marked tumor reductions. Severe and prolonged bone marrow depression caused the death of about one third ofour patients, a complication usually prevented by autologous bone marrow infusion. MATERIALS AND METHODS A large dose of melphalan was given intravenously to 23 patients with advanced refractory myeloma (Table 1). Lower doses of 80 mg/m2 (one 74-year-old patient) and 100 mg/m2 (I 5 patients) were given as a single 30-minute infusion. When the limited efficacy and severe toxicity of this program were appreciated, a higher dose of 140 mg/m2 was delivered in two daily fractions followed by the infusion of 12 to 16 x l0 autologous marrow cells one day later (seven patients). Autologous bone marrow had been stored during VAD-induced second remissions in six patients and following inef- fective VAD treatment in one patient with only extramedullary disease. Bone marrow was collected under general anesthesia, frozen at 1 #{176}C/miannd kept at - 196 #{176fo}rC 4 to 27 months. CFU-GM of the stored marrows ranged from eight to 92 colonies per l0 nucleated cells (median, 2 1 ). Plasma cell counts ranged from 2% to 7% (median, 3%). All patients were admitted to the hospital, and seven were treated in a protected environment unit under strict gnotobiotic conditions. Antibiotic prophylaxis was performed with trimethoprim-sulfamethoxazole and ketoconazole. Patients were From the University of Texas System Cancer Center, Anderson Hospital and Tumor institute, Houston. Submitted Oct 29, /985; accepted Dec 1 7. 1985. Supported by Grant (`.437/6/ from the National Cancer tute and the Cu/len Trust Foundation. Address reprint requests to Dr Bart Barlogie. Department Hematology, 6723 Bertner Aye, Box 55, Houston, TX 77030. (c) 1 986 by Grune & Stratton, inc. 0006-497//86/6705-O0/8$03.00/0 M.D. Instiof previously refractory disease indicated that apparent drug resistance could be overcome by dose escalation. How- ever. short remission times in most responding patients were consistent with rapid regrowth of primordial tumor cells with high proliferative activity. Although high-dose melphalan was of limited benefit to patients with refrac- tory myeloma, further studies are necessary to clarify its role during earlier phases of disease. a 1986 by Grune & Stratton. Inc. advised of procedures and attendant risks in accordance with institutional guidelines and gave informed consent. The median age of patients treated with melphalan alone was 44 years, whereas the age of those who also received autologous marrow was 63 years. All patients were resistant to multiple prior chemotherapy regimens, and all but two were refractory to salvage VAD therapy.' Ten patients had never responded to any prior treatment, and 13 were relapsing to usually two prior regimens. All but three had a high or intermediate tumor mass by standard staging criteria. Flow cytometric (FCM) analyses of bone marrow samples for DNA and RNA content of plasma cells were conducted as described previously.3 Unfavorable cytometric features included the presence of hypodiploid and biclonal DNA stemlines and/or a RNA content of <4 times that of normal lymphocytes.4 With any of these features, only 20% of previously untreated patients and less than 10% of patients with resistant myeloma had responded to VAD."5 Ten of our patients had such unfavorable FCM features, previously recognized by multivaniate regression analysis to represent the single most important variable both for initial remission6 and to response to salvage treatment.' Serial studies were conducted to monitor antitumor effect and toxicity. A significant response was defined by a reduction in tumor mass (serum myeloma protein) exceeding 75% with the disappearance of Bence Jones protein excretion for at least 2 months.7 Five patients died within 6 weeks (early deaths), four of them with a marked antitumor effect comparable to that observed in responding patients. Relapse-free and overall survival times were calculated by life table analyses from the onset of treatment. RESULTS Ten of 23 patients who received high-dose melphalan responded for at least 2 months; four other patients respond- ed, but were rated as failures because they died between 4 and 6 weeks from infection due to neutropenia (Table 2). Tumor-halving times calculated from changes in serum myeloma protein levels were very short in all responding patients (median, 0.3 months), with a tumor mass reduction in four patients to levels lower than those observed after any prior therapy. All patients considered responsive showed clearing of bone marrow plasma cells (<7%). Patients with or without response had similar features in terms of tumor mass and immunoglobulin type. Likewise, the incidence of response was not affected by treatment conditions such as the protective environment or autologous bone marrow trans- plantation. Although only one of I 3 patients with unfavora- ble FCM features had responded to VAD previously, seven of ten patients with such abnormalities achieved a marked cytoreduction with high-dose melphalan (P < .01 , chi-square test) (Table 2). Similarly, five of ten previously unresponsive 1298 Blood. Vol 67, No 5 (May), 1986: pp 1298-1301 HIGH-DOSE MELPHALAN IN REFRACTORY MYELOMA 1299 Table 1 . High-Dose Melphala n in Refractory Myeloma Characteristic N Age (yr) Range Median Treated in protected environment No. prior treatment regimens Range Median Prior VAD salvage treatment No prior response Unfavorable Intermediate FCM features and high tumor mass Autologous Bone Marrow Support No Yes 16 7 24-74 44 4 35-66 63 3 1-7 3-6 33 14 7 82 92 14 6 Total 23 24-74 54 7 1-7 3 21 10 11 20 Treatment outcome Response Early death#{176} Failuret 6 4 10 4 15 62 8 Abbreviations: FCM, flow cytometric. `At 6 weeks from high dose melphalan. tSurvived >6 weeks from high-dose tumor mass reduction. melphalan and achieved <75% patients experienced a marked antitumor effect with high- dose melphalan in contrast with a 20% response rate for similar patients receiving VAD. The median survival of all patients was 4 months (Fig 1), and the median relapse-free survival of our ten responders was 4 months (range, 2.5 to 18+ months). Four patients are still alive (3, 6, 13, 20 months), including two patients who received bone marrow support. The major complication was marked and prolonged agranulocytosis and severe thrombocytopenia to less than l0,000/sL for a median of4 weeks that required a median of 40 units of transfused platelets per patient. With bone marrow transplantation and despite a higher melphalan dose, minimal leukocyte (but not granulocyte or platelet) recovery to at least 200/L proceeded significantly faster and more uniformly than in the other 16 patients not receiving bone marrow support (P < .05, Wilcoxon test) (Fig 2). Further hematologic recovery with granulocytes greater than l,500/.tL and platelets greater than 150,000/zL occurred in I 1 and seven of 16 patients without marrow support and in six and three of seven patients who received autologous marrow. As a result of severe and persistent neutropenia in all 16 Table 2. Response to High-Dose Melphalan According to Pretreatment Features . Prior Response Nucelic Ac id Features N Favorable Unfavorable Total Percentage Yes 13 5/8 1(4)/5 6(9)/13 46(69) No 10 1(2)/4 3/6 4(5)/10 40(50) Total Percentage 23 6(7)/12 100 50(58) 4(7)/li 36(64) 10(14)/23 43(61) Parentheses tion. include patients who died with >75% tumor cytoreduc- C 0 0 0 0. 0.40 0.20 No.of Pabsno Tot De 23 19 0 16 14 A 7 50 Ovetol W,SOUBIUT WithOUT Non fiNn 6 12 18 Months Fig 1 . Kaplan-Meier survival plots in patients with refractory myeloma receiving high-dose melphalan with (0) or without () autologous bone marrow. The melphalan dose was higher in the marrow support group (140 mg/rn2) compared with patients not receiving autografts. There was only one of seven drug-related deaths in the marrow transplant group compared with six of 16 among the remaining patients (see also Table 3). Four patients remain alive at 3. 6. 1 3. and 20 months. patients without marrow support, four patients with a marked antitumor effect and one patient with resistant myeloma died ofdisseminated fungal infection (Table 3). An additional patient, still in complete remission of his myelo- ma, died of Escherichia co/i sepsis resulting from secondary marrow aplasia after previous complete hematologic recov- ery. The high incidence of suspected or proven fungal infection led to amphotericin B treatment in 1 1 of 16 patients. In contrast, the only death among the seven patients e U 0. 0 0. 0 20 40 60 0 Days 20 40 60 Fig 2. Peripheral blood leukocyte recovery with and without bone marrow transplantation following high-dose melphalan treat- ment for refractory myeloma (for further details. see text and Fig 1 ). Even though the time course to complete hematologic recovery was similar in both groups, patients receiving marrow support demonstrated a faster and more uniform leukocyte (but not granulocyte or platelet) recovery to 200 and 500/tL. preventing death from neutropenic sepsis in all but one transplant recipient. 1300 BARLOGIE ET AL TabI e 3. High-D ose M elphala n in Refractory Myeloma Marrow Support No Antitumor Effect Yes No Causes of Death N Alive Melphalan-Related Proessive Disease 10 1 61 5 1 4 4 Total 16 2 6 8 Yes Total Yes No 41 31 72 0 1 1 3 1 4 receiving marrow infusion occurred in a patient with agranu- locytosis persisting beyond day 35 after treatment who died of Pseudomonas sepsis. This patient had received prior high-dose cyclophosphamide on two occasions prior to mar- row storage. Thus, although their median age was 20 years less, six of seven treatment-related deaths occurred among the patients who did not receive bone marrow support (Table 3). Other toxicities included reversible hemorrhagic episodes in eight, diarrhea in 1 3, and pancreatitis in two patients. DISCUSSION The median survival of patients with multiple myeloma is approximately 30 months, with both the pretreatment tumor load and the sensitivity to chemotherapy representing the major prognostic factors.8 Until recently, the likelihood of controlling myeloma resistant to melphalan-prednisone has been less than 20%. Although about 50% of the patients responded to the VAD regimen,' those who remain unrespon- sive or relapse to this program present a difficult problem. The observation by McElwain and Powles that a high dose of melphalan induced a marked degree of remission in all nine patients including five who were resistant to standard mel- phalan seemed promising.2 We evaluated this program in a group of patients who were older, had received extensive prior chemotherapy including doxorubicin, and had cytomet- nc features associated with resistance to treatment."5 Considering the adverse prognostic features, the rapid and marked tumor cytoreduction in 61% of 23 patients confirmed remarkable antitumor activity from a single course of mel- phalan at a dose only three to four times higher than that given with standard therapy. Responses occurred even in patients who had been refractory to all prior therapies and with plasma cell nucleic acid features associated with drug resistance."5 Thus, as with the VAD regimen, the activity of high-dose melphalan confirmed that primary drug resistance could be overcome by dose escalation.2 Although myeloma protein reductions were rapid and marked, the remission durations were short. If one considers tumor-halving time as an index of drug sensitivity, shorter halving times should be associated with longer resmissions. However, we observed the paradox of progressive shortening of both tumor-halving time (medians of 1.5, 0.5, and 0.3 months) and remission duration (medians of 22, 1 2 and 4 months) from successive treatments (initial chemotherapy, first salvage VAD, and high-dose melphalan, respectively). These findings were compatible with the progressive expan- sion of more proliferative primordial tumor cells during the disease course. The high-dose melphalan program was more toxic than any treatment evaluated previously in patients with multiple myeloma. The 31% mortality rate in patients who did not receive bone marrow support was explained by the high incidence of infection during marked and prolonged neutro- penia. This complication was alleviated by autologous bone marrow support in older patients who received even higher doses of melphalan. The comparable degree and speed of response after high-dose melphalan with or without marrow support suggested that marrow contamination by well-differ- entiated tumor cells did not compromise tumor cytoreduc- tion. Persistent thrombocytopenia of 50 to 75,000/sL has been observed in other autologous marrow transplantation programs, but usually in conjunction with considerably higher doses of cytotoxic drugs. Our patients had extensive prior therapy and marrow involvement by tumor cells, either of which might have contributed to the reduced functional capacity of transplanted normal cells. The short remission duration should discourage further use of this highly toxic treatment in patients with resistant myeloma. The observation by McElwain and Powles of durable remissions in previously untreated patients, however, deserves further exploration.2"0 We favor such high-dose therapy approach, perhaps in conjunction with total-body irradiation, for consolidation in remission. The availability of autologous marrow stored early in remission would facilitate host support, and selection of patients with chemotherapy- sensitive myeloma should enhance the probability of attain- ing marked tumor cytoreduction and prolonged disease control. The recent demonstration of common acute lympho- blastic leukemia antigen-positive plasma cell precursors and of plasma cell-specific antigens offers the possibility of in vitro monoclonal antibody treatment for preferential tumor cell removal prior to marrow ACKNOWLEDGMENT The authors gratefully acknowledge Kay Delasalle and Leslie Smallwood for their help in analyzing clinical and laboratory data and Mattie Scott-Thomas for her secretarial support. REFERENCES I . Barlogie B, Smith L, Alexanian advanced multiple myeloma refractory EngI J Med 310:1353-1356, 1984 R: Effective to alkylating treatment of agents. New 2. McElwain Ti, Powles RL: High-dose intravenous melphalan for plasma-cell leukaemia and myeloma. Lancet 822-824, 1983 Ploidy and proliferative characteristics in monoclonal gammopa- thies. Blood 59:43-51, 1982 4. Smith L, Barlogie B, Alexanian R: Biclonal and hypodiploid multiple myeloma. Am J Med 1986 (in press) 5. Barlogie B, Alexanian R, Smith L, Dixon D, Smallwood, L, Delasalle K: Prognostic implications of tumor cell DNA and RNA 3. Latreille J, Barlogie B, Johnston D, Drewinko B, Alexanian R: content in multiple myeloma. Blood 66:338-341, 1985 HIGH-DOSE MELPHALAN IN REFRACTORY MYELOMA 1301 6. Barlogie B, Alexanian R, Gehan E, Smallwood L, Smith T, Drewinko B: Marrow cytometry and prognosis in myeloma. i Clin Invest 72:853-861, 1983 7. McLaughlin P, Alexanian R: Myeloma protein kinetics following chemotherapy. Blood 60:851-855, 1982 8. Alexanian R, Balcerzak 5, Bonnet i, Gehan E, Haut A, Hewlett J, Monto R: Prognostic factors in multiple myeloma. Cancer 36:1 192-1202, 1975 ,9. Bonnet J, Alexanian R, Salmon 5, Bottomley R, Amare M, Haut A, Dixon D: Vincristine, BCNU, doxorubicin and prednisone (VBAP) combination in the treatment of relapsing or resistant myeloma. Cancer Treat Rep 66: 1267-1 271 I 982 10. McElwain Ti: Personal communication. 1985 1 1 . Caliganis-Cappio F, ianossy G, Bergui L, Tesio L, Pizzolo G, Malavasi F, Chilosi M, Campana D, van Camp B, Gavosto F: Identification of malignant plasma cell precursors in the bone marrow ofmultiple myeloma. i Clin Invest 76:1243-1251, 1985 I 2. Ruiz-Arguelles Gi, Katzmann JA, Greipp PR, Gonchoroff Ni, Garton iP, Kyle RA: Multiple Myeloma: Circulating lympho- cytes that express plasma cell antigens. Blood 64:352-356, 1984 13. Anderson KC, Bates MP, Slaughenhoupt B, Schlossman SF, Nadler LM: A monoclonal antibody with reactivity restricted to normal and neoplastic plasma cells. J Immunol 132:3172-3179, I 984