Evaluation of clinical scale CD34 cell purification: experience of 71 immunoaffinity column procedures

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1 Bone Marrow Transplantation, (1997) 20, Stockton Press All rights reserved /97 $12.00 Evaluation of clinical scale CD34 cell purification: experience of 71 immunoaffinity column procedures MJ Watts, AM Sullivan, SJ Ings, D Leverett, AJ Peniket, AR Perry, CD Williams, S Devereux, AH Goldstone and DC Linch University College London Medical School and University College London Hospitals, London, UK Summary: ensure rapid neutrophil and platelet recovery and defined the minimum requirements as /kg, below which Seventy-one mobilised PBSC collections were subject to there is a very high risk of delayed or failed platelet CD34 cell purification using the CEPRATE SC stem engraftment. Either whole peripheral blood stem cells may cell concentration system. The overall median purity of be infused or the progenitors can first be purified. Such CD34 cells was 69% (6 93%). CD34 cell, and GM- purification procedures serve to purge the collection of CFC recoveries were 52% (8 107%) and 36% (3 contaminating tumour cells, 13 and are a prerequisite for in 118%). Purity was logarithmically related to the input vitro stem cell expansion and gene therapy. 14,15 The use of percentage of CD34 cells and starting requirements monoclonal antibodies to purify progenitors was first were established of 1% CD34 cell content for optimal described over 15 years ago, 16 with the initial studies using purity and a minimum of /kg CD34 cells to negative selection. Subsequently, positive selection with ensure recovery of our minimum engraftment threshold antibodies to the CD34 antigen was developed and has of /kg CD34 cells. Reduction of the washing proved amenable to the scaling up required for clinical steps reduced non-specific cell losses and shortened the practice. Several clinical scale devices are now commercially procedure but did not affect progenitor cell recovery. available. Important issues are the purity of the Purified CD34 cells were reinfused following high-dose enriched fraction, the cell losses that occur and whether therapy in 35 patients. The median time to neutrophil such losses frequently prevent the attainment of the desired recovery of /l was 12 (10 23) days and to the progenitor thresholds. The time required for the processing attainment of platelet independence was 13 (7 100) procedure is an important logistical consideration. This days. The risks of delayed platelet recovery were related report describes our experience with the CEPRATE system to the CD34 cell dose infused and were identical to the risks when non-purified PBSC collections were used. In conclusion, purification of CD34 cells using the CEPR- ATE device is reliable and the purified product results in prompt engraftment. The cell losses that occur do however restrict its use in many patients. Keywords: CD34 + cell; purification; regeneration on 71 apheresis collections from 56 individuals mobilised for PBSC (50 patients with lymphoma or myeloma and six normal donors). Materials and methods Patient groups Twenty-three patients with relapsed or resistant lymphoma, 27 myeloma patients and six normal donors were studied. High-dose chemo/radiotherapy with autologous stem cell All CD34 purification procedures were performed on a sinsupport has been shown to be of value in a number of gle apheresis harvest processed within 1 h of collection. In malignant disorders. 1 5 Peripheral blood stem cell trans- 15 subjects, 2 consecutive days apheresis collections were plantation (PBSCT) has largely replaced bone marrow processed giving a total of 71 CEPRATE (CellPro, Bothell, transplantation in the autologous setting (ABMT) because WA, USA) column procedures. The patient details and PBSCT results in a more rapid haematological recovery CD34 purification procedures performed are shown in with an attendant reduction in resource utilisation. 6 9 There Table 1. is a considerable consensus that there is a threshold requirement for peripheral blood progenitor cells assessed either by CD34 + enumeration or by GM-CFC In a study of Conditioning 81 previously treated lymphoma patients all treated on the The ablative therapy used for the lymphoma patients was high-dose BEAM chemotherapy regimen, we found that the the BEAM protocol 1,3 (BCNU 300 mg/m 2 i.v. on day 1, optimal number of CD34 + cells was /kg to etoposide 200 mg/m 2 /day i.v. days 2 5, (total dose 800 mg/m 2 ) cytosine arabinoside 200 mg/m 2 twice a day on days 2 5 (total dose 1600 mg/m 2 ) and melphalan 140 Correspondence: Dr DC Linch, The Department of Haematology, Univermg/m 2 i.v. on day 6. The ablative therapy for the myeloma sity College London Medical School, 98, Chenies Mews, London WC1E 6HX, UK patients was 100 to 140 mg/m 2 melphalan and 8 to 12 Gy Received 25 January 1997; accepted 27 April 1997 of fractionated total body irradiation.

2 158 Table 1 Evaluation of CD34 cell purification Subjects studied and number of CD34 + cell purification procedures performed Diagnosis Patient No. Age Sex Paired CD34 Total No. CD34 median (range) M:F procedures procedures Myeloma (37 65) 20: HD 7 30 (17 48) 3:4 1 8 HGNHL (20 63) 10: Normal donors 6 40 (33 63) 3: No. CEPRATE procedures HD = Hodgkin s disease; HGNHL = high-grade non-hodgkin s lymphoma. PBSC mobilisation and collection PBSC were collected on a continuous flow cell separator (COBE Spectra, COBE Laboratories Ltd, Gloucester, UK, or Baxter CS3000, Baxter Healthcare Ltd, Northampton, UK). The six normal donors were mobilised with G-CSF (filgrastim) at 10 g/kg/day s.c. and apheresis collections performed on day 5 and 6. Four of the lymphoma patients were mobilised with G-CSF beginning 6 days after ESHAP chemotherapy (etoposide 40 mg/m 2 for 4 days, methylpred- nisolone 500 mg daily, cytarabine 2 g/m 2 and cisplatin 25 mg/m 2 over 4 days). A single vial of G-CSF (lenograstim 263 g/vial) was given daily from completion of ESHAP until harvesting was complete. Apheresis commenced from day 15 when the recovery WBC first exceeded /l. The remaining patients were mobilised with a protocol described previously. 6 Low-dose cyclophosphamide (1.5 g/m 2 ) was given on the first day (day 1) followed by G- CSF given s.c. at 10 g/kg (filgrastim) or a single vial of lenograstim (263 g/vial) 24 h afterwards and daily there- after until harvesting was complete. Two to three apheresis collections were performed on days 8 to 12 commencing when the recovery WBC first exceeded /l. At least one unmanipulated apheresis collection was cryopreserved and held in reserve for each patient. Purified CD34 + cell cryopreservation In the first protocol modification (no pre-moab wash) 3 mg of the CD34 antibody was added directly to the harvest without prior PBS washing in a final incubation volume of between 60 ml and 110 ml and the procedure thereafter was as in the standard protocol. Omission of this pre-wash step reduced the processing time from around 2 h to 1. h. In the second modification (no wash method) both the prewash and the post-antibody PBS wash steps were omitted. Only 1 mg of the CD34 antibody was used in this protocol to prevent saturation of the avidin bead column. Following the antibody incubation step the harvest was made up to a final 300 ml volume in PBS containing 10 U/ml of heparin and the product loaded on the column. CD34 + selected cells were obtained from the apheresis product within 1 h with this protocol. In all cases the CD34 + selection process resulted in an approximate 90 ml cell fraction which was concentrated by centrifugation to 4.5 ml and an equal volume of tissue cul- ture grade PBS (Dulbecco s phosphate-buffered saline without calcium or magnesium; Sigma, Dorset, UK), con- taining 15% DMSO and 8% human serum albumin added as cryopreservative. The cells were then transferred to two 5 ml cryopreservative vials (Nalgene, Rochester, NY, USA) and left at 80 C overnight in a controlled rate freezing device (Nalgene, Cryo 1 C freezing container). The follow- ing day the vials were quickly transferred to sterile 15-ml polypropylene tubes (Becton Dickinson, Franklin Lakes, NY, USA), secured in a cardboard folder and stored in liquid nitrogen. cell processor (30 min). Three milligrammes (1 mg/ml) of the supplied biotinylated CD34 monoclonal antibody was added to the harvest for a 25-min period at room temperature in a final incubation volume of 75 ml. (The standard protocol has since been revised from 3 mg antibody in ml to 3 mg in a 150 ml volume.) The two wash steps with 1 l of PBS were repeated after the incubation period to remove unbound antibody (30 min). The washed CD34 antibody-labelled cells were then made up to a final volume of 300 ml in PBS and connected to the CEPRATE device. The column run stage of the procedure is automated and takes 35 min. As the cells flow through the device, avidincoated polyacrylamide beads within the immunoaffinity column capture the biotin moiety of the CD34-labelled cells allowing the unadsorbed CD34 negative cells to run through to a waste bag. The final part of the program is mechanical agitation and rinse of the capture beads with PBS, releasing the bound CD34 + cells (adsorbed cell fraction) into a collection bag in an approximate 90 ml volume. Overall the standard procedure takes 2 h. Processing modifications Progenitor evaluation In all cases samples were removed for white cell and progenitor Stem cell purification counts from the harvest product, the antibody-lab- elled harvest as loaded on the column and from unadsorbed Using the standard CEPRATE protocol the apheresis product and adsorbed (CD34 + cell purified) fractions. CD34 + cells was washed twice with 1 l of tissue culture grade phos- were measured by flow cytometry as described previously 21 phate-buffered saline (PBS; Sigma) using a COBE 2991 with matched irrelevant control or directly conjugated

3 Evaluation of CD34 cell purification CD34-phycoerythrin monoclonal antibody (HPCA-2PE; viously defined as the minimum target to ensure prompt Becton Dickinson). Cytocentrifuge preparations were prepared haematological engraftment. 12 from the CD34 + cell-purified fraction in each pro- Most notably there was a wide range of purities and cedure and stained with May Grünwald Giemsa for blast yields obtained. This in part reflects the variable cellular cell morphology as a rapid guide to column performance. A input from different harvests. A logarithmic relationship second cytospin was stained for CD34 + cells using alkaline was observed between harvest CD34 concentration and phosphatase-anti-alkaline phosphatase (APAAP) immuno- resultant purity in the CD34 enriched fraction (Figure 1). enzymatic staining as previously described. 6 In those patients in whom the percentage of CD34 + cells Granulocyte monocyte colony-forming cells (GM-CFC) in the PBSC collection was 1% (n = 17) the median and burst-forming units erythroid (BFU-E) were simultaneously CD34 + cell dose prior to processing was /kg and evaluated in a methylcellulose based clonogenic the purity of the CD34 + cell fraction was 46% with a assay medium as previously described. 12 Harvest PBSC median dose in the final fraction of /kg. Only 6/17 cells were plated in triplicate at concentrations of (35%) of final fractions contained the minimum requirement /ml, the CD34 unadsorbed cell fraction at of /kg (Table 2). In the remaining 54 individ /ml and purified CD34 + cells at /ml and uals with 1% CD34 + cells in the PBSC collection the /ml. Colonies were counted after 14 days incubation median input dose was /kg and the final CD34 + at 37 C in a humidified CO 2 atmosphere. cell purity was 76% with a median dose in the final fraction of /kg. Fifty-two out of 54 (96%) of procedures Calculations used resulted in a final yield of /kg CD34 + cells. The following calculations were made for progenitor recovery (yields) and overall WBC losses. CD34 recovery % = Harvest CD Analysis of the CD34 + cells input in absolute cell numbers gives similar data. Of those 12 individuals in whom the starting material contained /kg only 4/12 procedures resulted in an adequate number of CD34 + cells in the final fraction. In those patients with /kg CD34 + Purified CD34 + cells cells in the starting material this was 54/59 (92%). 159 Harvest GM-CFC 10 5 GM-CFC recovery % = GM-CFC in purified CD34 + cells Overall WBC losses % = 100 Harvest WBC 10 9 Assessment of yield variability WBC recovered from column Although cell purity was closely related to CD34 + cell input, the CD34 + cell yield was highly variable (Figure 2) Results and unpredictable. To assess whether this was due to technical variation or was patient-specific, nine patients had Progenitor cell purity and recovery (yields) PBSC purified and processed identically on consecutive days. Input cell numbers were similar on each day for the Seventy-one CD34 selection procedures were performed on paired harvests, the median starting dose and percentage of 56 subjects. Overall the median purity of the CD34 + cell CD34 + cells was /kg and 1.3% and /kg enriched fraction was 69% with 52% recovery of the startbility but the yields on the 2 days in individual patients and 1.2%. There was again marked inter-individual varia- ing harvest CD34 + cells (Table 2). The losses of CD34 + cells were comparable to the overall white cell losses which were similar (Figure 3). were approximately 50% if the standard processing protocol procedure was employed. The recovery of GM-CFC overall was slighty less than for CD34 + cells at 36%. The Effects of the pre-column washing steps CD34 + cells obtained correlated with the input CD34 + cell There were considerable non-specific cell losses associated number (r = 0.796) and the median yield from a single with the recommended full procedure and in an effort to column procedure was /kg. In 51/71 instances reduce these, and thus hopefully improve the final CD34 + (82%) /kg were obtained which we have pre- cell yield, modifications were made to remove one or both Table 2 Effect of harvest concentration of CD34 + cells on subsequent purity and yield of CD34 + cell enriched fraction (median/range) Harvest values CD34 purified fraction (%) CD34 level CD34% CD /kg CD34 purity CD34 yield CD /kg GM-CFC yield All (n = 71) ( ) ( ) (6 93) (8 107) (58/71) (3 118) 1% (n = 54) ( ) ( ) (40 93) (10 107) (52/54) (5 118) 1% (n = 17) ( ) ( ) (6 71) (8 101) (6/17) (3 78)

4 160 Table 3 Evaluation of CD34 cell purification Outcome of CD34 purification with two modifications to standard CEPRATE processing (median/range) Processing Harvest values CD34 purified fraction Non-specific protocol WBC losses CD34% CD /kg CD34 purity CD34 yield GM-CFC yield WBC processing + column losses Standard % 40% 23% 50% (n = 21) ( ) ( ) (6 89) (8 102) (3 118) (13 75) No pre-moab wash % 52% 41% 34% (n = 20) ( ) ( ) (6 93) (14 107) (9 88) (0 84) No-wash % 53% 39% 1% (n = 30) ( ) ( ) (40.92) (10 106) (5 106) ( 1 30) 100 r = 0.71 (log fit) % Purity of CD34 selected cells % % yield/purity Figure 3 Similar results in CD34 + cell yield and purity from nine con- secutive PBSC collections processed identically Apheresis harvest CD34% Figure 1 Logarithmic increase in CD34 + cell enriched fraction purity as harvest CD34 + cell concentration is increased. 0 day 1 day 2 day 1 day 2 CD34+ cell yield P = 0.582* *paired t-test CD34+ cell purity P = 0.418* Patient numbers < % CD34+ cell recovery from harvest Figure 2 Distribution of CD34 + cell yields from PBSC harvests following CD34 + cell selection. of the preparative washing steps. The standard protocol was performed in 21 patients and included washing the harvest cells twice in 1 l PBS before addition of the CD34 antibody followed by a second PBS wash after the incubation period. To reduce non-specific cell loss during centrifugation, the antibody was added direct to the apheresis product in 20 cases. This resulted in a reduction in total cell losses from 50 to 34%. To further minimise non-specific WBC losses a final cohort of 30 apheresis products was processed without any pre-column wash steps (no-wash method). Nonspecific white cell losses were reduced further but the purity and yield of CD34 + cells were similar with all three processing methods (Table 3). The protocol modifications although not improving CD34 + cell yield did result in a worthwhile time saving from 2 h of 1 h. To futher ensure that these modifications were not deleterious to the purified CD34 + cell yield two consecutive apheresis collections were processed in a further six patients comparing PBS wash and no-wash methods. The starting dose of CD34 + cells was similar on both days as

5 Evaluation of CD34 cell purification was the percentage of CD34 + cells in the starting material, recovery was rapid at 12 and 13 days for neutrophil and the median values being /kg and 1.3% in the collections platelet recovery, respectively. subjected to the PBS wash procedure and /kg and 1.6% in the collections with the no-wash method. The total cell losses were significantly reduced Discussion from 48% with the PBS wash procedure and 11% with the This large experience with the CEPRATE purification no-wash method (P = ). As with the larger cohort device confirms that it is reliable and easy to use in the however the final CD34 + cell yield and purity were similar, clinical setting. The overall CD34 + cell purity and yield in being /kg and 66% respectively with the PBS wash all 71 CEPRATE procedures of 69 and 52% compares with products and /kg and 67% respectively with PBS values of 42 and 52% reported by Shpall et al 22 in 18 wash steps omitted. There was again considerable interindividual patients, 61 and 73% as reported by Brugger et al 23 in 21 variation but no statistically significant differ- patients and 47 and 77% as reported by Schiller et al 24 in ences were obtained in CD34 + cell yields and purities on 37 patients. The median losses of CD34 + cells in these ser- the 2 separate days. ies during purification have ranged from 27 to 53% but in individual cases the losses can be considerably greater. Such losses have an impact on the clinical use of the purified Haematological recovery after infusion of purified CD34 + products. We have previously defined the minimum cells CD34 + cell dose for PBSC transplantation as /kg, and to allow for the losses associated with purification at Seven of the 50 lymphoma and myeloma patients have not least /kg CD34 + cells are required prior to purifiyet had their purified CD34 + cells returned, one patient died cation. In a series of 97 pre-treated lymphoma patients of disease progression and a further patient died at day +2 mobilised with cyclophosphamide and G-CSF 12 only 51 of BEAM chemotherapy and is not evaluable. patients (53%) achieved this target with a single apheresis. Thirty-seven have had their purified CD34 + cells returned This means that if CD34 + cell purification is to have wider alone after BEAM chemotherapy (n = 17) or mel- use two or more purification procedures must be performed phalan/tbi (n = 20). The median time to recovery of neutrophils on consecutive apheresis collections or the collections of /l was 12 days (range 9 23 days) with pooled and processed on the same column. In the 97 only two patients having slow recovery defined as greater patients referred to above, pooling of two collections than 21 days. Seventeen patients received G-CSF from 6 allowed achievement of the /kg CD34 + cell threshold days after the infusion of purified CD34 + cells and the in an additional 20 patients (21%). Twenty-six percent median time to neutrophil recovery in these patients was of patients still remain below this threshold, although it is 12 days (range days) compared to 14.5 days (range likely that this number would be less if patient progenitor days) in the remainder (P = 0.05). The median time cells were mobilised and collected at an earlier stage of the to attainment of platelet independence, defined as a sustained, disease. The use of two or more CD34 selections per patient unsupported platelet count above /l without raises financial constraints, encouraging most centres to bleeding, was 13 days (range 7 100) with G-CSF administration pool collections. Some users have reported poor cell having no effect. Three patients had slow platelet recoveries associated with the processing of stored PBSC recovery (22, 26 and 27 days) and four patients had delayed products however, and a study is in progress to determine recovery at 32, 36, 41 and 100 days. The incidence of optimal storage conditions. delayed platelet recovery 4/37 (11%) is the same as we A noteworthy finding was that the purity of the final frac- have published previously (8%) in a series of 75 lymphoma tion achieved was closely related to the input CD34 + cell patients who received unmanipulated PBSC. 12 The four percentage. A starting percentage of 1% was found to be patients with delayed platelet recovery after infusion of associated with a high probability that the final purity CD34 + cell-purified PBSC received 3.1, 1.1, 1.6 and would be 60% (44/54 cases). The percentage of CD /kg CD34 cells which was in each case below our cells in the starting collection is of course related to the previously defined optimal threshold of /kg. 12 The absolute number of CD34 + cells in the harvest and in gen- one case that took 100 days to become platelet-independent eral a 1% threshold relates to an absolute threshold of about received less than our minimal threshold number of /kg which is required from the viewpoint of 50% /kg CD34 + cells. losses. In practice therefore it is possible to abort a CD34 + In the final four patients the purified CD34 + cell dose cell purification before cell losses and unnecessary expense was considered inadequate to return alone at 0.6, 0.3, 0.3 have been incurred. Appreciation of the relationship and /kg and in each case two unmanipulated between input and output will also be important when back-up PBSC collections were returned concomitantly. In comparing the performance of different purification three patients even the pooled total dose of CD34 + cells devices. From a clinical standpoint input cells must refer collected remained low at 0.8, 0.6 and /kg and a to harvest collection values. A statement of final purity and bone marrow harvest was collected in the poorest mobiliser. yield without input CD34 + percentages is not meaningful. Slow neutrophil recovery was seen in two of these patients In this study, we have evaluated the impact of reducing at 26, 24 and 12 days and delayed platelet recovery in two the centrifugation/wash steps to try and minimise the cell at 18, 59 and 80 days, respectively. In the fourth patient the two pooled back up PBSC collections were above the minimum threshold level at /kg CD34 + cells and losses. The non-specific losses can be significantly ameliorated by such modifications but unfortunately the specific CD34 + cell losses are not reduced. 161

6 Evaluation of CD34 cell purification 162 The fact that haematological regeneration after purified 8 Schmidt N, Linch DC, Dreger P et al. Randomised trial of CD34 + cell transplantation was not delayed relative to filgrastim mobilised peripheral blood progenitor cell transplant whole PBSC is in accord with our previous data in a vs autologous bone-marrow transplantation in lymphoma smaller number of cases. 21 This implies that accessory cells patients. Lancet 1996; 347: are not required for rapid haematological recovery even 9 Smith TJ, Hillner BE, Schmitz N et al. Economic analysis of a randomized clinical trial to compare filgrastim-mobilized when no post-infusion growth factors are given. Identical peripheral blood transplantation and autologous bone marrow haematological recovery with CD34-purified or unsepartransplantation in patients with Hodgkin s and non-hodgkin s ated PBSC has also been reported by Brugger et al. 23 It lymphoma. J Clin Oncol 1997; 15: should be noted however that CD34 + cell losses do occur 10 Demirer T, Besinger WI. Optimization of peripheral blood with purification and if threshold limits are not applied stem cell collection. Curr Opin Haematol 1995; 2: before and after purification, an increase in delayed platelet 11 Bensinger W, Appelbaum F, Rowley S et al. Factors that recovery would be anticipated. influence collection and engraftment of autologous peripheral In conclusion, the CEPRATE device is a reliable system blood stem cells. J Clin Oncol 1995; 13: for purification of CD34 + cells on a clinical scale. The 12 Watts MJ, Sullivan AM, Jamieson E et al. Progenitor cell results in terms of purity of the final fraction are closely mobilisation after low dose cyclophosphamide and G-CSF: an related to the input CD34 + cell count. It is a relatively rapid analysis of progenitor cell quantity and quality and factors pre- process and can be futher speeded up by omission of recmalignant dicting for these parameters in 101 pretreated patients with ommended wash steps, although this does not improve cell lymphoma. J Clin Oncol 1997; 15: yield. The CD34 + cell losses are approximately 50% and 13 Cagnoni PJ, Shpall EJ. Mobilization and selection of CD34- are a significant problem in many cases, preventing the positve hemopoietic progenitors. Blood Rev 1996; 10: Williams SF, Lee WJ, Bender JG et al. Selection and expanattainment of the CD34 + cell thresholds required to ensure sion of peripheral blood CD34 + cells in autologous stem cell rapid haematological engraftment. transplantation for breast cancer. Blood 1996; 87: Brenner MK. Gene transfer into human hematopoietic progenitor cells: a review of current clinical protocols. J Hematother Acknowledgements 1993; 2: Beverley PCL, Linch DC, Delia D et al. Isolation of haematopoietic progenitor cells using monoclonal antibodies. Nature AR Perry is supported by the Leukaemia Research Fund, AJ Peni- 1980; 287: ket by Nexstar Ltd, UK and CD Williams by Roche Ltd UK. 17 Civin CI, Strauss LC, Brovall C et al. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG- 1a cells. J Immunol 1984; 133: Berenson RJ, Besinger WI, Hill RS et al. Engraftment after References infusion of CD34 + marrow cells in patients with breast cancer or neuroblastoma. Blood 1991; 77: Linch DC, Winfield D, Goldstone AH et al. Dose intensifi- 19 Maroleau JP, Brice P, Dal Cortivo L et al. CD34 + selection cation with autologous bone marrow transplantation is effec- by immunomagnetic selection (Isolex 300) for patients with tive in relapsed and resistant Hodgkin s Disease: results of a malignancies. Blood 1996; 88 (Suppl. 1) 110a (Abstr. 427). BNLI randomized trial. Lancet 1993; 341: Richel D, Johnsen H, Canon J et al. Highly purified CD Philip T, Guglielmi C, Hagenbeek A et al. Autologous bone cells isolated with the Amgen cell selector provide rapid marrow transplantation as compared with salvage chemo- engraftment following high-dose chemotherapy in breast cantherapy in relapses of chemotherapy-sensitive non-hodgkin s cer patients. Blood 1996; 88 (Suppl. 1): 110a (Abstr. 429). lymphoma. New Engl J Med 1995; 333: Watts MJ, Jones HM, Sullivan AM et al. Accessory cells do 3 Haioun C, Lepage E, Gisselbrecht Ch et al. Autologous bone not contribute to G-CSF or IL-6 production nor to rapid marrow transplantation (ABMT) vs sequential chemotherapy haematological recovery following peripheral blood stem cell for aggressive Non-Hodgkin s Lymphoma (NHL) in first transplantation. Br J Haematol 1995; 91: remission (CR): a study of 542 patients (LNH87 2 protocol). 22 Shpall EJ, Jones RB, Bearman SI et al. Transplantation of Blood 1995; 86 (Suppl. 1): 457a (Abstr. 1815). enriched CD34-positive autologous marrow into breast cancer 4 Attal M, Harousseau JL, Stoppa AM et al. A prospective, ranpatients following high-dose chemotherapy: influence of domized trial of autologous bone marrow transplantation and CD34-positive progenitors and growth factors on engraftment. chemotherapy in multiple myeloma. New Engl J Med 1996; J Clin Oncol 1994; 12: : Brugger W, Henschler R, Heimfeld S et al. Positively selected 5 Bezwoda WR, Seymour L, Dansey RD. High dose chemoautologous CD34 + cells and unseparated peripheral blood protherapy with hematopoietic rescue as primary treatment for genitor cells mediate identical hematopoietic engraftment after metastatic breast cancer: a randomised trial. J Clin Oncol high-dose VP-16, ifosfamide, carboplatin, and epirubicin. 1995; 13: Jones HM, Jones SA, Watts MJ et al. Development of a sim- Blood 1994; 84: plified single apheresis approach for peripheral blood progeni- 24 Schiller G, Vescio R, Freytes C et al. Transplantation of tor cell transplantation in previously treated patients with lymchemotherapy for patients with advanced multiple myeloma. CD34 + peripheral blood progenitor cells after high-dose phoma. J Clin Oncol 1994; 12: Beyer J, Schwella N, Zingsem J et al. Hematopoietic rescue Blood 1995; 86: after high-dose chemotherapy using autologous peripheralblood progenitor cells or bone marrow: a randomized comparison. J Clin Oncol 1995; 13: