Experimental Hematology 28 (2000) Service d Hématologie de l Hôpital Edouard Herriot, Lyon, France; b

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1 Experimental Hematology 28 (2000) Transplantation with selected autologous peripheral blood hematopoietic stem cells (HSCs) in multiple myeloma: Impact of HSC dose on engraftment, safety, and immune reconstitution Mauricette Michallet a, Thierry Philip b, Irène Philip b, Hubert Godinot c, Catherine Sebban b, Gilles Salles d, Anne Thiebaut a, Pierre Biron b, Francis Lopez c, Philippe Mazars c, Nora Roubi c, Tom Leemhuis c, Elie Hanania c, Christopher Reading c, Gilbert Fine c, Kerry Atkinson c, Chris Juttner c, Bertrand Coiffier d, Denis Fière a, and Eric Archimbaud a, a Service d Hématologie de l Hôpital Edouard Herriot, Lyon, France; b Service de Médecine du Centre Léon Bérard, Lyon, France; c SyStemix International, Lyon, France and SyStemix Incorporated, Palo Alto, Calif., USA; and d Service d Hématologie du Centre Hospitalier, Lyon-Sud, France (Received 30 December 1999; revised 25 February 2000; accepted 8 March 2000) Objective. The aims of our study performed in myeloma were to evaluate the performance and the safety of Systemix s high-speed clinical cell sorter, to assess the safety and efficacy of deescalating cell dose cohorts of hematopoietic stem cells (HSCs) as autologous grafts by determining engraftment, and to assess the residual tumor cell contamination using polymerase chain reaction (PCR) amplification assays of patient-specific complementarity determining region III (CDR III) analysis for residual myeloma cells. Materials and Methods. The clinical trial was performed in 31 multiple myeloma patients, using purified human HSCs mobilized from peripheral blood with cyclosphosphamide and granulocyte-macrophage colony-stimulating factor to support a single transplant after high-dose melphalan 140 mg/m 2 alone (cohort 1) and with total body irradiation (TBI) (cohorts 2 5) after an HSC transplant cell dose de-escalation/escalation design. Results. Twenty-three patients were transplanted. Engraftment data in the melphalan TBI cohorts confirmed that HSC doses above the threshold dose of HSCs/ kg provided prompt engraftment (absolute neutrophil count /L day 10; platelet count /L day 13). A higher rate of infections was observed in the early and late follow-up phases than usually reported after CD34 selected or unselected autologous transplantation, which did not correlate with the HSC dose infused. Successful PCR for CDR III could only be performed in five patients on initial apheresis product and final HSC product and showed a median tumor log reduction Conclusions. HSCs are markedly depleted or free of detectable tumor cells in multiple myeloma and are capable of producing fast and durable hematopoietic reconstitution at cell doses HSCs/kg. The delayed immune reconstitution observed is not different from that described in unselected autologous bone marrow and peripheral blood mononucleated cells transplants in multiple myeloma and may be corrected by addition of T cells either to the graft or to the patient in the posttransplant phase International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: CD34 Thy1 hematopoietic stem cell autologous transplantation Multiple myeloma Offprint requests to: Mauricette Michallet, Unité de Greffe de Cellules Souches Hématopoiétiques, Hôpital Edouard Herriot, Place d Arsonval, Lyon cedex 03, France; mauricette.michallet@chu-lyon.fr Deceased. Introduction High-dose cytotoxic chemotherapy and/or radiotherapy followed by transplantation of either unselected or selected autologous peripheral blood mononuclear cells (PBMCs) increasingly is being used to treat patients with hematologic malignancies and solid tumors. In some of those cases, tumor cells may be circulating in the blood at the time of collection [1 5]. In other patients, they may be mobilized out of the marrow into the blood stream by the mobilization regimen [6]. Several gene-marking studies showed that such X/00 $ see front matter. Copyright 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S X(00)

2 M. Michallet et al./experimental Hematology 28 (2000) malignant cells may contribute to posttransplant relapse in patients with acute myeloid leukemia, chronic myeloid leukemia, and neuroblastoma [7 9]. For neuroblastoma, retroviral-mediated gene-marking techniques showed that residual tumor cells in marrow have considerable clonogenic potential in vivo [9]. Multiple myeloma is consistently fatal when treated with conventional therapy alone [10]. Myeloablative therapy followed by autologous transplantation of unselected stem cells prolongs disease-free and overall survival [11], although more than 70% of patients eventually relapsed. Most unselected autografts are contaminated to a variable degree by myeloma cells [12]. Using quantitative polymerase chain reaction (PCR) amplification assays of patient-specific complementarity determining region III (CDR III) DNA sequences from bone marrow (BM) and PBMC samples, myeloma cells have been detected in nearly all patients [13,14]. Most patients have between 0.1% and 1% clonal B cells in their PBMC collections [15], resulting in the infusion of 0.5 to myeloma cells, which may contribute to relapse after transplant. In an attempt to obtain tumor-free grafts, BM of myeloma patients has been purged using chemical [16] and immunologic [17] techniques, but these purging techniques led to delayed engraftment. Stem cell selection based on antigens not expressed by myeloma cells might reduce plasma cell contamination [18,19], but with the available CD34 selection techniques used in various hematologic malignancies and solid tumors, the CD34 purity of such preparations varies between 30% and 90% [20 23]. The lack of purity of such preparations in multiple myeloma would leave detectable levels of myeloma cells in the graft [15,22]. Although controversial, some studies indicate that cells expressing the CD34 antigen are clonal in multiple myeloma patients [24,25]. This observation might reduce the potential clinical value of cell selection based on CD34 alone in multiple myeloma and indicates that an additional step may be necessary to achieve significantly more tumor depletion in the graft [15,22]. purified hematopoietic stem cells (HSCs) constitute a subpopulation (40 60%) of CD34 cells in mobilized peripheral blood. These HSCs can produce hematopoietic reconstitution in vivo in animal models [26 28]. Moreover, some studies suggested that the antigen combination might not be expressed by the most immature multiple myeloma cells [24,25]. Based on these data, a multicenter clinical study to mobilize HSCs from PBMC in support of high-dose chemotherapy/radiotherapy regimens in multiple myeloma patients was performed. The aims were to evaluate the performance and the safety of the purification device, to assess the safety and efficacy of de-escalating cell dose cohorts of HSCs as autologous grafts by determining the kinetics and extent of hematopoietic and lymphoid engraftment, and to assess the residual tumor cell contamination using PCR CDR III analysis for residual myeloma cells. Patients and methods Patients Patients with symptomatic multiple myeloma were screened in the three participating transplant centers in Lyon. To be enrolled in this study, patients had to receive first-line chemotherapy with vincristine, Adriamycin, and dexamethasone (VAD) [29,30] and to achieve at least a partial response (PR) after a minimum of three courses. Thirty-one patients (18 women and 13 men; median age 55 years, range 24 65) were enrolled in the study between March 1996 and January Characteristics at diagnosis According to the Durie and Salmon classification [31], 24 patients (77%) were stage III (16 IIIA, 8 IIIB) and 7 stage IIA. Thirty patients had a myeloma M component: 7 patients only secreted light chains in the urine and 23 had a serum M component: 13 IgG (6 lambda and 7 kappa), 7 IgA (2 lambda, 5 kappa), 2 IgD lambda, and 1 patient had a double IgG kappa and IgA kappa component. One patient presented with a nonsecreting myeloma. Sixteen patients had Bence-Jones proteinuria. Twenty-two patients (71%) presented with bone lesions that were associated with hypercalcemia in 10 cases. Eight patients had renal insufficiency and 20 presented with anemia. The median percentage of plasma cells in BM aspirates was 50% (range 6 100%), and the median value of beta-2 microglobulin was 3.5 mg/l (range ). Characteristics at enrollment All patients were in PR after having received a median of 4 courses (range 3 6) of first-line chemotherapy with VAD. The median interval between first chemotherapy and mobilization chemotherapy was 4.5 months (range 3 8.5), and the median delay between last course of chemotherapy and mobilization was 37 days (range 29 76). All patients had 60% myeloma cells in their BM aspirate and a normal blood count at time of mobilization. Creatinine level was below 150 mol/l, and patients had normal hepatic, cardiac (systolic ejection fraction 50%), and pulmonary function. All patients had negative serology for human immunodeficiency viruses (HIV1 and 2), human T lymphocyte viruses (HTLV1 and 2), hepatitis C virus, and hepatitis B surface antigen and had no evidence of active infection. Twenty-three patients (74%) were seropositive for cytomegalovirus (CMV) at screening. All patients signed an ethics committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale) approved informed consent. Trial design This trial was an open-label, single-arm, cell dose de-escalation/ escalation trial evaluating autologous transplantation of varying numbers of HSC purified from PBMCs (Fig. 1). Target HSC doses at the time of cryopreservation were determined by cohort, using the ideal body weight for the first patient and the actual body weight at time of mobilization in the remaining patients. The trial originally contained four cell dose cohorts. The cell doses were chosen based on preclinical results and on the limited transplant experience with cells acquired by the Arkansas team [32] at the time our study was initiated. For safety reasons as a result of the uncertainty of engraftment with this new source and production method of putatively more primitive and highly selected cells, the first patients enrolled

3 860 M. Michallet et al./experimental Hematology 28 (2000) in this trial received a nonmyeloablative regimen with only melphalan 140 mg/m 2. Progression to each subsequent cohort was based on the demonstration of sustained engraftment with adequate safety in 2 of 3 (cohorts 1 and 2) or 4 of 5 (cohort 3) evaluable patients in the cohort currently being studied. Patients in cohort 1 (n 3) were to receive melphalan alone 140 mg/m 2 (day 2) followed by reinfusion of HSCs/kg 10% on day 0. The protocol was amended after the first patient experienced delayed engraftment. Subsequent patients of that cohort received HSCs/kg. Patients in cohorts 2 to 4 were to receive fractionated total body irradiation (TBI) of 8 Gy (days 7 to 4) and melphalan 140 mg/m 2 (day 2), followed on day 0 by reinfusion of HSCs/kg (n 4), equal to HSCs/kg 10% (n 5), and equal to HSCs/kg 10% (n 5) for cohorts 2 to 4, respectively. After considering the engraftment results in cohorts 2 to 4, a fifth cohort was added, using the same conditioning regimen (melphalan TBI) to infuse additional patients with 0.7 to HSCs/kg (n 6). Addition of this cohort aimed to see if a plateau in the time to early engraftment was reached by using the higher doses. Trial objectives The primary objectives of this study were to evaluate the safety of the reinfusion of HSCs, to assess the performance and safety of the high speed clinical cell sorter, and to assess the efficacy of HSCs by determining the kinetics and extent of hematopoietic engraftment after infusion of an autologous HSC graft. The secondary objectives of this trial were to evaluate the safety and efficiency of the mobilization regimen to stimulate sufficient numbers of progenitor cells to obtain a minimum infusion product of HSCs/kg 10%, to assess the characteristics of autologous HSC grafts including cell number, purity, and time to engraftment, to explore the minimum HSC dose required for engraftment and to assess the residual tumor cell contamination of the selected HSCs using PCR CDR III analysis for the presence of residual myeloma cells. Mobilization regimen PBMC mobilization was attained by the administration of 6g/m 2 cyclophosphamide followed by daily subcutaneous injections of 250 g/m 2 granulocyte-macrophage colony-stimulating factor Figure 1. Schema of study design. Patients were mobilized with granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclophosphamide. Peripheral blood mononuclear cells (PBMC) were collected and cells selected using a two-step process. For safety concerns with this new source of cells, the first cohort of patient recieved melphalan alone as conditioning regimen followed by infusion. Subsequent patient cohorts received melphalan total body irradiation (TBI) and decreasing numbers of cells. Progression from cohort 1 to 4 was based on the demonstration of sustained engraftment and adequate safety in the current cohort. The fifth cohort was added after considering poor engraftment results with the low cell dose cohorts. All patients received G-CSF after transplant and were followed-up 1 year after transplant.

4 M. Michallet et al./experimental Hematology 28 (2000) (GM-CSF) (Leucomax, Schering-Plough) until completion of the collections. Leukapheresis was begun when the white blood cell (WBC) count was at least /L and the platelet count was at least /L, increasing independently of transfusion. The apheresis product was diluted in a SyStemix proprietary shipping medium and delivered to SyStemix Lyon for processing provided that (for technical reasons associated with the cell sorting technology) the percentage of HSC was 1.5% at the mid-point of the first apheresis (first 29 patients), or that the first apheresis contained at least CD34 cells/kg (two last patients). Additional apheresis products were sent to SyStemix for processing until the target HSC dose was cryopreserved. After obtaining a sufficient number of HSCs for transplantation, additional PBMC collections were performed in all patients to obtain an unselected backup graft containing at least CD34 cells/kg. Stem cell purification Either immediately on receipt or after an overnight hold at room temperature, SyStemix personnel performed one of two different methods for purifying HSC. The HSCs were all ultimately purified by flow cytometric cell sorting. However, the presort enrichment steps for the first 29 patient samples used a combination of counterflow centrifugal elutriation [33] and chemical depletion using phenylalanine methylester (PME) (Terumo Medical Corp, Elkton, MD), which eliminates mature monocytes, granulocytes, erythrocytes, and platelets by chemical lysis [32]. Because of technical evolution in the manufacturing process, the last two patient samples were preenriched for CD34 cells using the Isolex 300 SA (Baxter Immunotherapy) immunomagnetic positive selection device [34] before staining and sorting for HSCs. The HSCs were stained and sorted as previously described [32] using the SyStemix proprietary dual-laser, fluorescence-activated, high-speed clinical cell sorter [35], at a rate of to events per second. HSCs were sorted directly into a culture tube containing a small volume of the SyStemix proprietary culture medium. All procedures were performed at SyStemix Lyon using standard operating procedures and in accordance with good manufacturing practices. Cryopreservation The cells were washed, counted with a hemocytometer, resuspended to a maximum concentration of cells/ml in a SyStemix-manufactured proprietary cryopreservation medium containing cryoprotectants, and aliquoted into at least two cryovials. Cells were frozen using a programmable step-down freezer with a freezing profile previously optimized for survival. After the product temperature reached 140 C, the vials were transferred to the liquid phase of a liquid nitrogen cryostorage tank at SyStemix Lyon. HSC doses were released for clinical use after endotoxin level ( 35 EU per infused dose), sterility (absence of bacterial and fungal organisms), and purity ( 80%) had been performed on the HSC infusion product (HSC-IP). The cryopreserved HSC vials were delivered to the Centre Léon Bérard (CLB) in a liquid nitrogen dry shipper just before the patients received their conditioning regimen and were immediately transferred back to the liquid phase of liquid nitrogen. Infusion After completion of the high-dose chemotherapy or chemoradiotherapy regimen, the HSC-IP was thawed in a 37 C waterbath at the Laboratoire de Greffes Hématopoïtiques of CLB. The cells were slowly diluted two times in SyStemix sterile formulation medium and transferred to a 30-ml syringe. A cell count with a trypan blue viability test was performed using a hemocytometer on a 100- L sample of the thawed product. As a result of previous human engraftment experience [32], a post thaw HSC viability 80% required immediate infusion of the backup unselected graft. HSC-IP was quickly delivered to the clinical unit for immediate infusion via intravenous push through the patient s central line. All efforts were made to ensure that less than 1 hour elapsed between thawing and infusion of the HSC-IP. All patients began subcutaneous granulocyte colony-stimulating factor (G-CSF) injections at a daily dose of 5 g/ kg 1 day after receiving the HSC-IP, until the absolute neutrophil count (ANC) was /L for 4 consecutive days and increasing on each of those 4 consecutive days. If the ANC dropped below /L, G-CSF was resumed at a daily dose of 5 g/kg. Engraftment evaluation Engraftment status was based on neutrophil and platelet counts. The key parameters were number of days to reach ANC /L while receiving G-CSF treatment, number of days to reach the last platelet transfusion given for a platelet count /L or for bleeding, and the number of days to reach a platelet count /L, independently of platelet transfusion. The number of days to reach a platelet count /L was not presented as a key parameter because it often is confounded by platelet transfusion noise. Patients were evaluated at day 14 by BM aspiration and biopsy for the presence of erythroid or myeloid precursors. BM examination was to be repeated in patients who did not achieve neutrophil engraftment by day 21 or day 28, and the absence of myeloid or erythroid engraftment in these patients would require infusion of the unselected backup graft. Response criteria Patients were evaluated for multiple myeloma response at days 100, 180, and 365 after autologous transplantation. A PR required a reduction of at least 50% in serum monoclonal component and a reduction of Bence-Jones proteinuria to 200 mg/day and to 10% of pretreatment values. A complete response (CR) was defined by the absence of monoclonal gammapathy in serum and urine, using immunofixation analysis, a BM adequately cellular ( 20%) with 5% plasma cells by immunostaining, and a normal serum calcium level. New lytic lesions in bone, hypercalcemia, or other new evidence of disease constituted relapse or progressive disease. Flow sorting of plasma cells Flow sorting of plasma cells and generation of the CDR III primer for allele-specific oligonucleotide (ASO). Enriched myeloma cells collected before transplant were used to generate patient-specific CDR III sequence. Synthesis and testing of each patient s ASO was performed using quantitative real-time PCR. Patient peripheral blood samples for minimal residual disease analysis were obtained before and after cell processing. BM samples were collected at the time of enrollment and at 100, 180, and 365 days after transplant. The BM samples collected before mobilization were enriched for CD38 / CD45 /lo cells using flow sorting to allow patient-specific ASO determination. Genomic DNA was isolated and concentration was determined using the picogreen dsdna quantitation kit and a luminescence spectrometer in allele specific previously reported [36]. Statistical analysis Significance of group differences in endpoints measured on a continuous scale (e.g., age and number of platelet transfusions) was

5 862 M. Michallet et al./experimental Hematology 28 (2000) tested using Student s t-test and Wilcoxon rank sum test. The hypothesis of no relation between HSC dose and CD3, CD4, CD8, and CD56 counts and immunoglobulin levels was analyzed using Spearman correlation. Group differences in binary and categoric endpoints (e.g., number of patients failing to engraft) were assessed using Fisher exact tests. All tests were two-sided and performed at the 5% level of significance. Four patients who failed to achieve platelet engraftment before terminating study participation (after 75, 112, 142, and beyond 365 days) were included as if they achieved this endpoint later than the highest observed time to engraftment (91 days) for evaluating group median values. Results Patient participation in each step of this clinical trial is summarized in Figure 1. Mobilization phase The first apheresis was performed at a median of 14 days (range 11 20) after beginning the mobilization regimen, and the product contained a median of CD34 cells/kg (range CD34 cells/kg). Among the first 29 patients who had their PBMC samples preprocessed by the elutriation and chemical depletion method, six failed to meet the 1.5% acceptance criterion at midpoint of the first apheresis for cell processing (median 0.33% HSC; range %) and dropped out of the trial. They had a median total of /kg CD34 cells collected for their backup graft (range /kg) in a median of three aphereses (range 2 4). Five of these six patients proceeded with intensive therapy supported by an unselected stem cell transplant. The last two patients who had preprocessing using the immunomagnetic positive selection method met the acceptance criteria. Twenty-five patients successfully mobilized (Table 1), including three patients who were processed despite low HSC levels (1.2%, 1.4%, and 1.4%) at midpoint of their first apheresis. The median percentage of HSC cells in the first apheresis product was 3.1% (range %), with a median HSC content of /kg (range /kg). A median of two aphereses per patient (range 1 4) was necessary to collect a median number of CD34 cells /kg (range /kg) with a median HSC content of these CD34 cells of 47.4% (range %). The overall HSC recovery after cell processing was 24.1% (range %), the median HSC purity of the final product was 90% (range 81 98%), and a median total number of viable HSC/kg (range /kg) was cryopreserved. There was considerable variability in the process yield that resulted from the technical complexity and individual inherent variability of the multiple procedures (e.g., counterflow centrifugation, chemical depletion with PME, and high-speed sorting) required to produce the HSC product. Twenty-nine of 48 apheresis products were analyzed for myeloma cells by flow cytometry and showed a median plasma cell content of 8% (range %). The backup graft was obtained in a median of one additional aphereses (range 0.5 4) and contained a median of CD34 cells/kg (range /kg). Stem cell purification CDR III analysis for the presence of myeloma-related clonal B cells was performed on 11 of the 25 successfully processed patients; five analyses were completed. Because most of the baseline BM samples analyzed were collected after first-line chemotherapy (VAD), the levels of plasma cells unique to the myeloma were very low and initial identification of the relevant CDR III clones was not possible for 6 of these 11 patients. This multistep assay required approximately 8 weeks to complete, and SyStemix resource limitations prevented performing this analysis in every patient. Among the five patients successfully analyzed, the median reduction in tumor burden between the apheresis and the cryopreserved selected cells was 3.12 log (range 2.29 to 4.12 log) (Table 2). HSC infusion Among the 25 patients who mobilized successfully, two subsequently developed complications and dropped out of the trial before autologous transplantation. The first patient (no. 3) developed acute renal failure thought to be secondary to progression of myeloma and was finally transplanted with an unselected PBMC graft; the second (no. 6) developed unexplained prolonged thrombocytopenia after mobilization and was not transplanted.twenty-three patients were infused with a median cryopreserved dose of HSCs/kg (range /kg). The median cell viability on thawing was 96% (range 84 99%), and patients received a median dose of pure and viable HSCs/kg (range /kg). Results for each cohort are given in Table 1. Transplant results Engraftment endpoints (ANC /L and platelet count /L) according to the HSC dose infused are shown in Figure 2. Preliminary analysis of other studies with HSCs suggested infusions above a threshold dose of HSCs/kg produced rapid neutrophil and platelet engraftment. Engraftment kinetic results by cohort are reported in Table 3 and are described later, separated according to the conditioning regimen received by the patients (melphalan TBI), and according to whether patients received above or below the threshold dose of HSCs/ kg for patients who received melphalan and TBI. All adverse events were graded according to the South West Oncology Group criteria. During the posttransplant period, patients received either single donor platelet infusions or multiple donor platelet infusions (seven platelet units).

6 M. Michallet et al./experimental Hematology 28 (2000) Table 1. Cell selection and characteristics of grafts Pt. no. Sex Delay last chemo MM mobil stage (days) (1) No. aph (2) CD34 (10 6 /kg) (3) in CD34 pop (%) (4) cryopr (10 6 /kg) (5) recov (%) (6) purity (%) (7) for transp (10 6 /kg) (8) viability (%) (9) infused (10 6 /kg) (10) Cohort 1 1 M IIA F IIIB * * * * 4 M IIA M IIIA M IIA * * * * Median values Cohort 2 7 F IIIA F IIIA F IIA F IIIA Median values Cohort 3 11 F IIIA M IIIB M IIIA F IIA M IIIA M IIIB Median values Cohort 4 19 F IIIA M IIIA M IIIB F IIIA Median values Cohort 5 23 F IIIB M IIIA F IIIB F IIIB F IIIA F IIIB Median values Overall population Median values* *Patients 3 and 6 were not infused with HSCs due to renal insufficiency before transplant and thrombocytopenia before transplant, respectively. (1) Number of days between the last chemotherapy and the mobilization chemotherapy; (2) number of aphereses performed for HSC selection (does not include aphereses performed for the backup graft); (3) total number of CD34 cells/kg collected; (4) percentage of HSC within the total CD34 cell population; (5) total number of HSC/kg cryopreserved; (6) HSC recovery after cell processing; (7) HSC purity of cryopreserved sample; (8) total number of HSC/kg used in transplant to match the cohort dose; (9) HSC viability after thawing; (10) total number of postthaw viable and pure HSC/kg infused. Engraftment kinetics and toxicity: melphalan regimen Three patients were infused in cohort 1. Patient 1 (dose HSCs/kg) demonstrated initial neutrophil engraftment with ANC /L on day 13. The patient resumed G-CSF therapy on day 22 due to decrease in ANC to /L and eventually became G-CSF independent on day 36. The patient reached a platelet count /L on day 57. Due to this unexpected delayed engraftment, the target HSC dose for this cohort was amended. Subsequent patients received HSCs/ kg and demonstrated satisfactory neutrophil and platelet engraftment (Table 3). During the initial hospitalization, patients 1, 4, and 5 received either single donor platelet infusions (14, 0, and 0, respectively) or multiple donor platelet infusions (1, 4, and 4, respectively). The equivalent number of platelet units transfused was 91, 28, and 28, respectively. During the immediate posttransplant period ( 30 days), two patients developed gram-positive coccal septicemia (with septic shock in the first patient), and one developed gram-positive coccal bacteremia and candidemia. During the late follow-up phase ( 30 days), one patient (no. 1) experienced a transverse longitudinal sinus thrombophlebitis on day 382.

7 864 M. Michallet et al./experimental Hematology 28 (2000) Table 2. Minimal residual disease CDR III evaluation Pt. 3 Pt. 4 Pt. 5 Pt. 7 Pt No. of processed aphereses Mean percent of CDR III positive cells Day 1 apheresis 4.000% 0.070% 0.044% 0.175% 0.600% Day 1 HSC product 0.010% DL DL DL 0.057% Day 2 apheresis 1.550% 0.100% 0.037% 0.067% 0.350% Day 2 HSC product 0.036% DL DL 0.057% Day 3 apheresis 0.260% 0.048% 0.550% Day 3 HSC product 0.005% DL 0.067% Pt. 3 Pt. 4 Pt. 5 Pt. 7 Pt. 8 Estimated tumor cells in apheresis sample (1) Estimated tumor cells in phsc cryopreserved sample (2) Average fold reduction in tumor burden Average log reduction in tumor burden Data in italics represent mean percent tumor cells (CDR III positive) of all available experimental data in the apheresis product and in the final cryopreserved sample. DL below the detection limit; no positive CDR III signal observed on the total number of cells used for that assay. (1) Estimated tumor cells in apheresis sample, based on the percentage of CDR III positive signal and on the total number of cells in the apheresis product; (2) conservative estimates of tumor cell content of HSC cryopreserved sample. Tumor cell content is based either on the percentage of CDR III positive signal or on the detection limit of specific assays and on the total number of cells in the cryopreserved product. Engraftment kinetics and toxicity: melphalan TBI regimen A total of 20 patients received the melphalan TBI conditioning regimen in cohorts 2 to 5. Results are presented according to the dose of HSC infused, above or below the potential threshold dose of /kg. The two groups were not statistically different for sex, age, disease stage, beta-2 microglobulin level at diagnosis, and delay between last chemotherapy and mobilization procedure. However, the mean purity of the final infusion product was lower in patients receiving less than the threshold dose of HSC/kg (87.3% 0.8% vs 92.5% 1.2%; p 0.002), and these patients had a significantly higher mean number of previous VAD courses (4.5 vs 3.5; p 0.012). The neutrophil and platelet engraftment kinetics according to the threshold dose infused are shown in Figure 2. Engraftment results, infectious complications, and multiple myeloma response are shown in Table 4. HSC dose /kg. Twelve patients (six men and 6 women) received HSCs/kg. They all initially reached an ANC /L in a median of 12 days (range days). Six (50%) patients (nos. 11, 13, 17, and 18 in cohort 3; no. 21 in cohort 4; no. 27 in cohort 5) resumed G-CSF therapy on at least one occasion due to a decrease in ANC to /L, and three eventually became G-CSF independent. Three patients remained G-CSF dependent and failed to show signs of platelet engraftment. This was associated with reactivated severe CMV infections requiring ganciclovir therapy in all three patients. The first patient (no. 13 in cohort 3, HSCs/kg) was reinfused with the unselected PBMC backup graft on day 142; the second (no. 21 in cohort 4, HSCs/kg) died on day 75 after transplant from severe sepsis; the third (no. 27 in cohort 5, HSCs/kg) developed pulmonary aspergillus infection finally leading to acute respiratory distress syndrome (ARDS) and death on day 112 after transplant. Another patient (no. 19 in cohort 4, HSCs/kg) had successful neutrophil engraftment, but failed to show platelet engraftment and remained platelet transfusion dependent 370 days after transplant. Among these 12 patients, 8 (67%) reached a platelet count /L, in a median of 35 days (range 16 91). In these eight patients, the last platelet transfusion was given at a median of 10.5 days (range 6 16) after transplant. During the initial hospitalization, patients received either single donor platelet infusions (median 2.5, range 0 34) or multiple donor platelet infusions (median 4, range 0 8). The median number of platelet units transfused was 51 (range ). Eleven of the 12 patients (91%) experienced a febrile episode during aplasia with microbiologic documentation of an organism in five patients (42%). Two (16%) pulmonary aspergillus and two herpes simplex infections occured in this patient group. Other adverse events included grade 2 to 3 mucositis (83%) and grade 3 biochemical liver toxicity (16%) that resolved during follow-up. During the late follow-up phase ( day 30), two of the 12 patients (16%) experienced severe septicemia with pseudomonas aeruginosa (nos. 21 and 13) on days 73 and 88, leading to ARDS and death on day 75 in 1 case (no. 21) and resolution in the other (no. 13). Both patients had an ANC count /L at the time of septicemia. There were three CMV infections requiring ganciclovir therapy, and six patients (50%) experienced herpes zoster infection (at days 32, 82, 129, 167, 175, and 176 after transplant). Other adverse

8 M. Michallet et al./experimental Hematology 28 (2000) follow-up phase ( day 30), there were 2 (16%) herpes zoster infections (at days 34 and 192 after transplant), one purulent tonsillitis, and three other infections. Two patients developed severe hemolytic syndromes. The first (no. 8) experienced hemolytic anemia on day 62 after transplant (positive direct Coombs test) whereas the second (no. 30) experienced a severe hemolytic uremic syndrome on day 133 after transplant. Both events resolved with appropriate therapy. One patient (no. 24) developed acute myeloid leukemia on day 446 after transplantation leading to uncontrolled sepsis and acute pulmonary edema, which resulted in death on day 457. Immune reconstitution: melphalan regimen (n 3) The median total lymphocyte count remained subnormal up to 6 months after transplant and reached normal values 1 year after transplant. Median CD3 T-cell count reached normal values on day 42, then fell and remained subnormal up to 1 year after transplantation. Median CD3 CD8 T-cell count reached normal values on day 21 and remained within normal values thereafter. Median CD3 CD4 T-cell count remained below normal values at 1 year after transplant. Kinetics of lymphocyte subsets are shown in Figure 3. Figure 2. Neutrophil and platelet engraftment after transplant (median values) for melphalan total body irradiation (TBI) regimen. Gray areas represent normal laboratory ranges. events included idiopathic thrombocytopenia on day 140 after transplant in one patient, which resolved under corticosteroid therapy, and fatal myocardial infarction on day 120 in one patient with a normal hemoglobin level. HSC dose /kg. Eight patients (one man and seven women) in cohorts 2 to 5 received HSCs/kg. They all initially reached an ANC /L in a median of 10 days (range days). Patient 30 (cohort 5, HSCs/kg) resumed G-CSF therapy on three occasions due to a decrease in ANC to /L and eventually became G-CSF independent. Platelet counts /L were reached in all patients in a median of 13 days (range 9 72). The last platelet transfusion was given at a median of 9 days (range 5 25) after transplant. During the initial hospitalization, patients received either single donor platelet infusions (median 0.5, range 0 5) or multiple donor platelet infusions (median 2.5, range 0 5). The median number of platelet units transfused was 28 (range 14 47). Seven of the 8 patients (88%) experienced a febrile episode during aplasia with microbiologic documentation of an organism in five patients (63%), and one had herpes simplex infection. Other adverse events included grade 2 to 4 mucositis (63%) and grade 3 biochemical liver toxicity (13%) that resolved during follow-up. During the late Immune reconstitution: melphalan TBI regimen (n 20) The median total lymphocyte count reached normal values on day 70 after transplantation, then fell and remained subnormal up to 1 year after transplant. Median CD3 T-cell count reached normal values on day 70, was normal on days 100 and 180, but was subnormal 1 year after transplantation. Median CD3 CD8 T-cell count reached normal values on day 42, and median CD56 (natural killer) cell count reached normal values on day 28. Both cell populations remained within normal values thereafter. CD56 cell count on day 70 was significantly associated with the number of HSCs infused (p 0.02; Spearman correlation). CD3 CD4 T-cell counts remained below normal values 1 year after transplant. Kinetics of lymphocyte subsets according to the dose of HSC infused are shown in Figure 3. With the exception of CD56 cells, T-cell immune reconstitution did not correlate with the dose of HSCs infused. Patient follow-up after transplant The median follow-up of this study is 15 months, with a maximum of 25 months after transplantation. Among the 23 patients who received HSC transplants, one patient (no. 21) died of severe sepsis and ARDS on day 75. The remaining 22 patients were evaluated for early disease response at day 100 after transplant. At this point, six patients achieved a CR and 16 patients achieved a PR. Among the six patients achieving CR at day 100, two died before 1 year after transplant. One (no. 27) died of ARDS on day 112 and one (no. 20) died of an unassociated myocardial infarction on day 120. Among the 16 patients achieving PR at day 100, one patient (no. 13) received a backup transplant on day 142 after showing lack of sustained engraftment and re-

9 866 M. Michallet et al./experimental Hematology 28 (2000) Table 3. Engraftment results and multiple myeloma response after transplantation Patient no. Day ANC /L (1) Day platelet /L (2) Day last platelet transf (3) Platelet transf (units) (4) MM resp (5) Status (6) Cohort CR Alive and well 24 mpt PR Alive and well 22 mpt PR Alive and well 21 mpt Median values Cohort PR Progression at 13 mpt PR Alive and well 19 mpt PR Progression at 11 mpt and death due to MM PR Alive and well 19 mpt Median values Cohort CR Alive and well 18 mpt NR NR 36 PR back-up graft infused on day 142, progression 6 mpbut PR Alive and well 16 mpt PR Alive and well 13 mpt PR Progression 12 mpt PR Progression 14 mpt Median values Cohort NR NR 20 CR Alive and well (but still plt transf dependent) CR Death from cardiac arrest on day 120 pt NR NR 46 NE Death from sepsis and ARDS on day 75 pt PR Progression 12 mpt Median values Cohort CR Alive and well 12 mpt PR Alive and well 12 mpt NR NR 49 CR Death from ARDS on day 112 pt PR Alive and well 8 mpt PR Alive and well 6 mpt PR Alive and well 5 mpt Median values Overall population Median values 11 28* 10* 20 CR complete response; mpbut month post backup tranpslant; mpt month post transplant; NE not evaluable; NR not reached; PR partial response; pt post transplant. (1) Number of days to reach an ANC count /L; (2) number of days to a platelet count /L; (3) days of the last platelet transfusion given for platelet count /L or for bleeding; (4) total number of platelet units transfused during the initial hospitalization period; (5) multiple myeloma response; (6) actual patient follow-up and status on July 31, *Median based on the population that successfully engrafted. lapsed 6 months after rescue, and three patients have not yet reached their 1-year visit. Sixteen patients were evaluable for disease response at 1 year after transplant. Four patients (nos. 1, 11, 19, and 23) showed sustained CR. Among the patients in PR at day 100 after transplant, three (nos. 9, 17, and 22) progressed (at 11, 12, and 12 months after transplant, respectively) and received second-line therapy, and nine remained in stable PR. Two other patients (nos. 7 and 18) progressed after 1 year after transplant (at 13 and 14 months after transplant, respectively) and received secondline therapy. BM evaluations showed decreased myeloma cell number over time. Cytologic analysis showed decreasing median numbers of plasma cells in the BM from 50% at diagnosis (range %; n 23) to 12.3% (range 1 60%; n 22) at screening, 5% (range 1 55%; n 18) pretransplant, 3.8% (range %; n 22) at day 100, and 3% (range 1 20%; n 12) at 1 year after transplant. CDR III analysis showed decreasing median numbers of CDR III positive cells from 2.97% (range %; n 4) at screening, to 1.5% (range %; n 4) at day 100 and 0.7% (range %; n 4) at 1 year after transplant. Clinical features (age, sex), prognostic criteria (multiple myeloma stage, beta-2 microglobulin at diagnosis), number of previous chemotherapy courses (VAD), and transplant parameters (con-

10 M. Michallet et al./experimental Hematology 28 (2000) Table 4. Engraftment, infectious complications, and multiple myeloma response for melphalan TBI regimen according to the dose of HSC infused (n 20) infused dose /kg /kg p Value Note Number of patients 12 8 Days post transplant to ANC /L 12 (10 13) 10 (10 11) c G-CSF resumed (no. of patients) 6 (50%) 1 (13%) a G-CSF dependence (no. of patients) 3 (25%) a Days post transplant to platelet count /L 64 (16 365) 13 (9 72) c Platelet engraftment failure (no. of patients) 4 (33%) a Days post transplant to last platelet transfusion 14 (6 365) 9 (5 25) c Infectious complications Febrile episodes ( day 30) 11 (91%) 7 (88%) a Septicemia ( day 30) 2 (16%) 2 (25%) a Pulmonary aspergillosis 2 (16%) a CMV infections 3 (25%) a Herpes zoster infections 6 (50%) 2 (25%) a Early response (day 100) Evaluable patients 11 8 CR 4 (36%) 1 (13%) a PR 7 (64%) 7 (87%) a Late response (1 year) Evaluable patients 8 5 CR 2 (25%) 1 (20%) a PR 4 (50%) 3 (60%) a Relapse 2 (25%) 1 (20%) a a Fisher exact test. Percentages are summarized. b Student s t-test. Mean (standard error) is summarized. c Wilcoxon rank sum test. Median (range) is summarized. ditioning regimen and HSC dose) did not impact on the multiple myeloma response rate in both early and longer-term follow-up after transplant, although no firm conclusions can be drawn due to the limited number of patients. Discussion High-dose therapy followed by autologous transplantation of unselected HSCs prolongs disease-free survival and overall survival in patients with aggressive multiple myeloma [11]. The number of infused cells that provides prompt, complete, and sustained engraftment after unselected PBMC transplantation has been determined to be CD34 cells/kg [37 39]. The same number of CD34 cells, selected with either an avidin-biotin immunoadsorption method [20 22,40 42] or an immunomagnetic positive selection method [43], also provides rapid hematologic recovery after transplant. The Thy1 subpopulation of CD34 cells have the self-renewal characteristics of HSCs and are able to generate all hematopoietic lineages [26 28], and this antigen combination is not expressed by the most immature multiple myeloma precursors [24,25], thus providing an opportunity to yield a tumor-free graft. A first report [32] showed that HSCs could be mobilized by cyclophosphamide and GM-CSF in multiple myeloma patients with less than 12 months of prior chemotherapy and could provide a highly tumor-depleted graft. However, Tricot s study with CD34 Thy1 HSCs/kg (median 0.46, range HSCs/kg) showed significantly delayed neutrophil and platelet engraftment compared to historical control unselected PBMC transplants after a first transplant and did not allow determination of a cell dose providing rapid and complete engraftment. The present study confirms that HSCs can be effectively mobilized with cyclophosphamide and GM- CSF in most patients (80%) and can be selected with SyStemix high-speed clinical cell sorter to reach a median purity of 90% (range 81 98%) in the infusion product. The present study shows that HSCs can be cryopreserved and are able to retain a high and homogeneous viability after thawing (median 96%, range 84 99%), better than previously reported by the Arkansas team (median 86%, range 51 93%) [32]. The wide range of cell doses ( HSC/kg) suggested that cell doses above a threshold dose of HSCs/kg provided rapid, complete, and sustained engraftment in most patients receiving melphalan TBI conditioning. Patients receiving less than this threshold dose experienced significantly delayed neutrophil (p 0.003) and platelet (p 0.008) engraftment compared to patients who received more than the threshold dose and engrafted similarly to patients receiving standard CD34 selected [41] or unselected [11,37]

11 868 M. Michallet et al./experimental Hematology 28 (2000) Figure 3. T-cell subset kinetics after transplant (median values). Gray areas represent normal laboratory ranges. autologous grafts. The delay to the last transfusion for a platelet count /L or bleeding also was greater in patients receiving less than the threshold dose and almost reached statistical significance (p 0.054). G-CSF requirements after initial engraftment, number of platelet engraftment failures, and number of platelet units transfused are greater in patients receiving less than the threshold dose, but these differences did not reach statistical difference. During the early follow-up phase, 91% of patients experienced febrile episodes (including septicemia [26%] and bacteremia [30%]), which is slightly higher than reported in other studies [22,32,42,44], although such high rates have been reported by others [45,46]. In the later follow-up phase of this study, there may have been an increased rate of viral infections. The three patients (13%) who reactivated CMV infection requiring ganciclovir therapy, and finally failed to engraft, all received lower doses of HSCs (0.31, 0.47, and /kg). Two of these patients (nos. 13 and 21) had proven reactivation of CMV infection in the mobilization phase of the trial and received ganciclovir therapy until just before transplantation, whereas the third patient (no. 27) experienced gram negative septicemia during the mobilization phase of the trial, possibly signifying an underlying immune deficiency in these patients. During the late follow-up there was an unexpectedly high rate (35%) of varicella-zoster virus infections, occuring at a median of 171 days after transplant (range ), and experienced by 50% of the patients having received HSCs/kg. Although there was no statistical difference in the infection rates between the two groups, the possible effect of the cell dose infused on immune reconstitution was analyzed. With the exception of CD56 cell level on day 70 after transplant, there was no significant difference in the kinetics of T-cell immune reconstitution according to the dose of HSC infused. The study shows subnormal total lymphocyte and CD3 cell counts, mainly related to a low CD3 CD4 cell count lasting for at least 1 year after transplantation. Abnormal phenotype and function of B and T cells after allogeneic or autologous transplantation with BM, PBMC, or selected CD34 cells have previously defined a diminished T-cell proliferative response and an imbalance in the CD4 /CD8 ratio [42,47 53]. This study also showed this decreased ratio, with CD3 CD8 reaching normal values rapidly after transplantation (day 42). There was rapid recovery of CD56 cell count (normal by day 28), thus confirming other reports showing that CD56 cell count and function rapidly recover after transplantation [54,55]. Abnormal cytokine production and T-cell receptor levels in the serum also have been reported [56 58]. Bomberger et al. [59] reported similar results and further focused on T-cell subset reconstitution, showing that circulating T cells were primarily memory T cells (CD45RO, CD45RA ) during the first 2 months after transplantation and mainly naive T cells thereafter (CD45RO, CD45RA ), suggesting a maturation of CD34 hematopoietic progenitors to naive T cells. The persistent distortion of the CD4 / CD8 ratio, coupled with the appearance of CD45RA naive T cells, suggests abnormal thymopoiesis in these patients. The decreased number of CD3 CD4 cells observed may not only reflect lack of thymic function but may result from the low T-cell content of HSC grafts. Some groups showed significantly faster immunologic reconstitution after PBMC transplantation compared to BM transplants [51], but this may have been influenced by the immune modifying effect of growth factors [51,60]. Quantitative recovery of peripheral lymphocytes has been reported to be delayed among recipients of CD34 selected HSC compared to conventional autografts [59]. In multiple myeloma, these findings were first confirmed in a small group of patients by Lemoli et al. [41]. More recently, the results of a randomized trial comparing unselected PBMC and avidin-biotin immunoadsorption selected CD34 transplantation in 131 multiple myeloma patients was reported and showed a low CD4 cell count at 1 year after transplant in both study arms, with a significant faster early CD4 recovery in the unselected arm [42]. The occurrence of autoimmune hemolytic syndrom