Progenitor Cell Mobilisation Optimising Parameters for Peripheral Blood Leukapheresis After R

Home , Ancestim

Bone Marrow Transplantation (2002) 30, 851–860  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt Progenitor cell mobilisation Optimising parameters for peripheral blood leukapheresis after r- metHuG-CSF (ﬁlgrastim) and r-metHuSCF (ancestim) in patients with multiple myeloma: a temporal analysis of CD34+ absolute counts and subsets

IH Chin-Yee1, M Keeney1, AK Stewart2, A Belch3, I Bence-Buckler4, S Couban5, K Howson-Jan1, M Rubinger6, D Stewart7, R Sutherland8, V Paragamian9, M Bhatia10 and R Foley 11

1London Health Sciences Centre, London, ON, Canada; 2Princess Margaret Hospital, Toronto, ON, Canada; 3Cross Cancer Institute, Edmonton, Alberta, Canada; 4Ottawa General Hospital, Ottawa, ON, Canada; 5Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia, Canada; 6Manitoba Cancer Treatment and Research Foundation, Winnipeg, Manitoba, Canada; 7Tom Baker Cancer Center, Calgary, Alberta, Canada; 8The Toronto Hospital, Toronto, ON, Canada; 9Amgen Canada, Mississauga, ON, Canada; 10Robarts Research Institute, London, ON, Canada; and 11Hamilton Health Sciences Centre, Hamilton, ON, Canada

Summary: and early progenitors and the ability to predictably schedule leukapheresis. Patients (n = 69) with multiple myeloma undergoing Bone Marrow Transplantation (2002) 30, 851–860. peripheral blood stem cell collection (PBSC) were doi:10.1038/sj.bmt.1703765 treated with cyclophosphamide and a combination of Keywords: multiple myeloma; CD34 subsets; SCF; G- recombinant methionyl human granulocyte colony-sti- CSF; stem cell mobilization mulating factor (r-metHuG-CSF, filgrastim) and recombinant methionyl human stem cell factor (r-metHuSCF, ancestim). The objectives of this study were to determine: (1) The proportion of patients reaching a target Results from several clinical trials using high-dose chemo- yield of у5 ؋ 106 CD34+ cells/kg in one or two success- therapy and hematopoietic stem cell transplantation from ive large-volume (20 liter) leukapheresis procedures; (2) either an autologous bone marrow1–3 or peripheral blood3– the optimal collection time for leukapheresis; (3) mobil- 5 have demonstrated a survival advantage for patients with ization kinetics of CD34+ subsets in response to G- multiple myeloma compared to results from trials using CSF/SCF. All patients were mobilized with cyclophos- conventional chemotherapy. Comparisons between PBSC phamide (2.5 g/m2) on day 0 followed by filgrastim (10 and autologous bone marrow transplantation (ABMT) in ␮g/kg ) plus ancestim (20 ␮g/kg) commencing day 1 and this patient population suggest a significantly shorter continuing to day 11 or 12. Of the 65 evaluable patients, median duration of neutropenia and superior remission 57 were considered not heavily pretreated and 96.5% rates and survival with PBSC transplantation compared obtained a target of у5 ؋ 106/kg in one collection. The with ABMT.6,7 median CD34+ cells/kg was 39.5 ؋ 106 (range: 5.2–221.2 Successful transplantation requires harvesting of an ؋ 106). Subset analysis demonstrated the number of adequate graft to ensure rapid and sustained recovery of CD38؊, CD33؊, and CD133+ peaked at day 11; and marrow function. Several studies have found a correlation CD34+, CD90+ cells peaked at day 10. The optimum day between the number of progenitor cells infused and the time for leukapheresis was determined to be day 11. The to hematopoietic reconstitution in patients with multiple median absolute peripheral blood CD34+ cell numbers myeloma.8–10 Chemotherapeutic agents including the use of on day 11 was 665 ؋ 106/l (range: 76–1481 ؋ 106/l). alkylators, such as melphalan, are known to adversely Eight of the 10 heavily pretreated patients were evalu- affect CD34+ progenitor/stem cell yield,8,11 and therefore able: three achieved the target dose in one leukapheresis may potentially exclude a large number of candidates who (37.5%) and three (37.5%) achieved the target dose with may otherwise benefit from PBSC transplantation. two leukaphereses. Use of this mobilization strategy Although a relationship between the rate of engraftment allowed the collection of high numbers of CD34+ cells and the yield of CD34+ cells has been demonstrated,8–10,12,13 others have suggested that the absolute yield of CD34+ cells may be less critical than the infusion of specific primitive subsets of CD34+ cells, eg CD34+ CD33Ϫ,14 CD34+ 38Ϫ,16 + + 17 Correspondence: Dr I Chin-Yee, London Health Sciences Centre, 800 and CD34 CD90 . Commissioners Rd East, London, Ontario, Canada, N6A 4G5 A number of strategies have been evaluated to improve Received 15 April 2002; accepted 30 July 2002 stem/progenitor cell mobilization and yield.18–25 Recombi- Filgrastim and ancestim in patients with MM IH Chin-Yee et al 852 nant methionyl human stem cell factor (r-metHuSCF, Study design ancestim), the natural ligand for c-kit, has been shown to This was a phase 2, open-label, multicenter study. The pri- act synergistically with a variety of other hematopoietic 26 mary endpoint was to determine the proportion of patients growth factors to increase colony formation in vitro. In who reached the target yield of у5 ϫ 106 CD34+ cells/kg preclinical models and clinical trials, synergy with filgras- in one or two successive large volume leukapheresis pro- tim enhances PBSC mobilization resulting in faster 27–30 cedures The secondary endpoints were to determine the engraftment at a lower cell dose. Synergy between fil- optimal timing for leukapheresis and to determine mobiliz- grastim and ancestim may increase not only the absolute + + ation kinetics of CD34 subsets in response to G-CSF/SCF. number of mobilized CD34 cells, but also the relative pro- The mobilization regimen consisted of cyclophosphamide portion of uncommitted stem cells and the optimal timing 2.5 g/m2 intravenously on day 0 followed by 20 ␮g/kg of apheresis.31,32 The objectives of this study were to assess у ancestim (Stemgen; Amgen Inc, Thousand Oaks, CA, the number of patients who achieved a target yield of 5 USA) plus 10 ␮g/kg filgrastim (Neupogen; Amgen Inc) ϫ 106 CD34+ cells/kg in one or two large-volume leukaph- + subcutaneously, from day 1 to completion of leukapheresis ereses and examine the kinetics of CD34 cell mobilization (day 11 or 12). Patients were premedicated with ranitidine in patients with multiple myeloma, using a combination of 150 mg, cetirizine 10 mg and salbutamol, two puffs before cyclophosphamide, filgrastim and ancestim to determine the administration of ancestim. Leukophereses was perfor- the optimal timing of leukapheresis. We also analyzed the med on days 11 and 12 (if target yield was not reached). temporal sequence of the appearance of specific subsets of + + Ϫ + Ϫ A blood volume of 20 l was processed on a COBE Spectra CD34 cells, including CD34 , CD38 , CD34 CD33 , or Baxter Fenwall CS3000 for each leukapheresis. CD34+ CD133+, and CD34+ CD90+ using a standard 33 Peripheral blood samples from day 0 and day 9 to day assay. 12, and leukapheresis samples from day 9 to day 12 were analyzed for CD34+ cells and CD34+ cell subsets. Leukaph- eresis samples were diluted 1:10 with phosphate buffered Patients and methods saline containing 10% fetal calf serum. CD34+ enumeration and subset analysis Patient population All samples were analyzed by a central laboratory using a The ethics committee for each of the participating insti- three-color, single-platform variant of the ISHAGE guide- tutions approved the protocol and all patients gave written lines. This procedure includes the addition of the viability informed consent before any study-related procedures were dye 7-amino actinomycin D (7-AAD),33,35,36 which allows done. Patients were eligible for study enrolment if they for the measurement of CD34+ cells directly from a flow were diagnosed with high-risk multiple myeloma (ie stage cytometer without the need for a hematology analyzer, and I with bone lesions and stage II or III according to the Durie a direct assessment of total leukocyte and CD34+ cell and Salmon classification34), were between 18 and 65 years viability in a single tube. Details of the methodology have of age, and had an Eastern Cooperative Oncology Group been previously described.33,36 In brief, StemKIT (ECOG) performance of 0 to 2. The initial accrual for this (Beckman-Coulter/Immunotech, Florida, USA) which uses study was limited to non-heavily pretreated patients, CD45FITC/CD34PE and FlowCOUNT fluorescent immu- defined as having received р six cycles of prior chemo- nospheres were used for determining absolute CD34+ therapy, with no more than two cycles containing mel- counts. Diluted peripheral blood or leukapheresis product phalan or nitrosoureas (BCNU). The study was sub- (100 ␮l) was added to 20 ␮l CD45FITC (clone sequently modified to include patients with more extensive J33)/CD34PE (clone 581) and 20 ␮l 7-AAD (final concen- chemotherapy exposure. Laboratory criteria included an tration 1 ␮g/ml). All analyses were performed in duplicate. absolute neutrophil count (ANC) of у1.5 ϫ 109/l, platelet Tubes were incubated for 15 min at room temperature and count у100 ϫ 109/l, serum creatinine р150 ␮mol/l; biliru- red blood cells were lysed with ammonium chloride for 10 bin, aspartate transferase (AST), and alanine aminotransfer- min (blood samples only). Leukapheresis samples, which ase (ALT) Ͻ twice times the upper limit of normal. contained few red blood cells, were suspended in phos- Subjects with MM in persistent relapse (not responsive phate-buffered saline. Samples were stored on ice until to second-line chemotherapy), who had a prior bone mar- analysis (within 1 h). No wash steps were used in the pro- row or PBPC transplant and those with a concurrent malig- cedure. Immediately before analysis, 100 ␮l FlowCOUNT nancy (other than treated basal cell carcinoma of the skin fluorospheres were added and tubes analyzed on a or cervical intra-epithelial neoplasia) were excluded. Due Beckman-Coulter XL-MCL flow cytometer (Figure 1). to the possibility of allergic type reactions to r-metHuSCF, Additional CD34+ subsets analyses of peripheral blood subjects with seasonal or recurrent asthma within the pre- and leukapheresis samples were done on samples from 20 vious 10 years, history of anaphylactic-type reactions to patients, using a three-color modified ISHAGE proto- food or insect bites, angioedema or recurrent urticaria were col.33,37 The subsets analyzed included CD38Ϫ (clone T16), also excluded. Other exclusion criteria were uncontrolled CD90+ (5E10), CD33Ϫ (03HL60.251), and CD133+ infection, significant comorbid conditions, pregnancy, bre- (AC133). All antibodies were gifts and provided from ast-feeding or allergy to Escherichia coli-derived products. Joelle Hirn (Immunotech, Marseille, France) with the Subjects receiving beta adrenergic blocking agents were exception of CD90, which was purchased (Pharmingen, also excluded. BDB, CA, USA).

Bone Marrow Transplantation Filgrastim and ancestim in patients with MM IH Chin-Yee et al 853 1023 1023 SS SS

B A E

00 100 101 102 103 100 101 102 103 CD45 FITC CD34 PE

1023 1023

CD34+ 250/ l SS SS D C

0 0 100 101 102 103 0 1023 CD45 FITC FS

103 F1 F2 1023

102

101 SS SS

F3 100 G

0 100 101 102 103 0 1023 CD45 FITC FS

103 1023 7-AAD+ 102 10% non-viable cells CAL

101 SS SS Flow count beads 100

0 0 0 1023 100 101 102 103 Time 7AAD

Figure 1 Histogram 1 shows all events with region A drawn to include all viable CD45+ leukocytes (excluding the dead 7-AAD+ events in region J, histogram 8). Histogram 2 is gated on viable CD45+ events (region A), and CD34+ events are selected (region B). In histogram 3, events from gates A and B are displayed and only those events that cluster are gated in region C. In histogram 4, events sequentially gated from A, B and C (viable CD34+ cells) are finally identified as those gated by a generic lymph-blast region D. Histogram 5 displays CD45 vs CD34 and histogram 6 shows FS vs SS of lymphocytes to allow a discriminant FALS to be set. Histogram 7 shows counting beads, selected by their bright fluorescence. Region CAL is placed on singlet beads. The sample contained 250 viable CD34+ cells/␮l.

Bone Marrow Transplantation Filgrastim and ancestim in patients with MM IH Chin-Yee et al 854 For the CD34+ subset three-color assay, CD34FITC and withdrawn from the study. A second patient developed CD45PC5 were combined with one of the following PE febrile neutropenia after cyclophosphamide administration labeled subsets: CD38, CD33, CD90, CD133. Diluted per- and did not proceed to leukapheresis. Of the other noneval- ipheral or leukapheresis products (100 ␮L) were added to uable patients, one sample was clotted and the fourth titrated antibodies and incubated for 20 min at room tem- patient was leukapheresed on days 13, 14 and 15 after mob- perature. Red blood cells were lysed with 2 ml ammonium ilization, off study protocol, and was not included in the chloride (as above, blood samples only) followed by two analysis, although the results will be discussed. All patients washes with PBS and suspension in PBS. A minimum of received at least two cycles (range, 2–8) of the VAD regi- 1000 CD34+ events were collected on each sample. Positive men (vincristine, adriamycin, dexamethasone) before PBSC analysis region was determined on autofluorescence of transplantation. In addition, 13 patients (19%) received gated CD34+ cells in FL2 channel (PE) on other chemotherapy regimens before VAD including CD34FITC/CD45PC5/unstained PE tube (Figure 2a, histo- melphalan/prednisone, cyclophosphamide, dexamethasone, gram 6). All subsequent tubes were analyzed at this cursor cyclophosphamide/etoposide/cisplatinum/dexamethasone, setting. Figure 2b shows representative histograms of the and CHOP (cyclophosphamide, doxorubicin, vincristine, four subsets gated on CD34+ cells as defined above. and prednisone). Nineteen patients (28%) received radiotherapy before PBSC transplantation. Statistical analysis Study endpoints Simple descriptive statistics were used to summarize, (1) The proportion of patients who achieved a target yield of The primary endpoint of the study was to determine the у5ϫ 106 CD34+ cells/kg in one or two large volume leu- proportion of patients who achieved a target yield of у5ϫ kapheresis; (2) The median peripheral blood CD34+ yield 106 CD34+ cells/kg in one or two large-volume leukapher- to determine optimal collection time for leukapheresis esis. Of the first 20 patients enrolled (one of whom was between day 9 and day 13; and (3) Analysis of the mobiliz- classified as heavily pretreated), all achieved the target of ation kinetics of CD34+ subsets CD38Ϫ, CD90+, CD33Ϫ, у5 ϫ 106 CD34+ cells/kg in a single large-volume leukaph- and CD133+. eresis. The median yield was 48.8 ϫ 106 CD34+ cells/kg in one collection (range, 9.1 to 221.2 ϫ 106/kg).

Results + CD34 cell yield Patient demographics and distribution Due to the increased number of CD34+ cells obtained in the first 20 subjects, eligibility criteria were expanded to A total of 69 patients from eight centers were enrolled in allow patients who had received extensive pretreatment this study, and 65 were evaluable. The baseline demo- before transplantation to enroll. In the second stage of the graphic and disease states are given in Table 1. The mobil- study, a further 49 patients were entered with a total of 10 ization regimen was well tolerated with the exception of patients being classified as heavily pretreated. Overall, a one patient who had an allergic reaction attributed to the total of 57 patients were evaluable in the non-heavily pre- administration of ancestim. This patient was subsequently treated group, with one patient not proceeding to leukapheresis due to an allergic reaction to SCF and one sample not Table 1 Patient demographics and baseline characteristics evaluable due to clotting. Of these 57 patients, 55 (96.5%) obtained a target of у5 ϫ 106 CD34+ cells/kg in one large- Sex (n/%) volume aphereses with two patients reaching the target in Men 42 (61%) two large-volume leukapheresis. Median CD34+ yield in Women 27 (39%) ϫ 6 + Age (years) the non-heavily pretreated group was 39.5 10 CD34 6 Median 56 cells/kg (range, 5.2–221.3 ϫ 10 ). Eight of the 10 patients Range 32–73 defined as heavily pretreated were evaluable. One patient Myeloma stage (n/%) did not proceed to leukapheresis due to the development of Stage I 3 (4.3%) febrile neutropenia after cyclophosphamide, and the other Stage II 20 (29%) Stage III 46 (66.7%) had leukaphereses on days 13, 14 and 15 off study protocol Duration of disease (months) and were therefore excluded from analysis. Three (37.5%) Median 5.5 patients achieved the target dose with one large-volume Range 2.8– 8.7 leukapheresis and three (37.5%) patients achieved the target Prior radiotherapy Number (%) 19 (28%) dose in two large-volume leukaphereses. The remaining 6 Prior chemotherapy two (25%) patients achieved a minimum of Ͼ 2 ϫ 10 + + Number (%) 69 (100%) CD34 cells/kg. Median CD34 yield in the heavily pre- Median number cycles 4 treated group was 5.7 ϫ 106 CD34+ cells/kg (range, 2.8– Range of number of cycles 2–48 25.3) (Table 2). Secondary endpoints included determi- Prior treatment with alkylators Number (%) 13 (19%) nation of optimal collection time for leukapheresis between Median number of cycles 5 days 9 and 13, and analysis of the mobilization kinetics of Range of number of cycles 3–48 CD34+ subsets CD38Ϫ, CD90+, CD33Ϫ and CD133+. The optimum day for leukapheresis, based on peripheral

Bone Marrow Transplantation Filgrastim and ancestim in patients with MM IH Chin-Yee et al 855 a (1) : Z0107300.LMD FL4 LOG/SS UnGated (1) : Z0107300.LMD FL1 LOG/SS A 1023 1023

SS 95.8% SS B 4.2%

E 0 6.8% 0 100 101 102 100 101 102 CD45PC5 CD45FITC

(1) : Z0107300.LMD FL4 LOG/SS A&B (1) : Z0107300.LMD FS/SS A&B&C 1023 1023 SS SS C D 98.9% 99.7%

0 0 100 101 102 0 1023 CD45PC5 FS

(1) : Z0107300.LMD FL2 LOG/SS E (1) : Z0107300.LMD FL2 LOG/FL1 LOG A&B&C&D 1023 G1 G2 H1 H2 0.0% 0.0% 99.2% 0.8% 102

SS 101 CD34FITC 100 H3 H4 0.0% 0.0% G3 G4 99.4% 0.6% 0 100 101 102 100 101 102 b Blank Blank (1) : Z0107306.LMD FL2 LOG/FL1 LOG abcd (1) : Z0107301.LMD FL2 LOG/FL1 LOG abcd H1 H2 H1 H2 102 0.3% 99.7% 102 88.9% 11.1%

101 101

100 H3 H4 100 H3 H4 0.0% 0.0% 0.0% 0.0%

100 101 102 100 101 102 CD38PE CD90PE (1) : Z0107302.LMD FL2 LOG/FL1 LOG abcd (1) : Z0107304.LMD FL2 LOG/FL1 LOG abcd H1 H2 H1 H2 102 35.8% 64.2% 102 25.2% 74.8%

101 101

100 H3 H4 CD34FITC100 H3 H4 CD34FITC 0.0% 0.0% 0.0% 0.0%

100 101 102 100 101 102 CD33PE AC133PE

Figure 2 (a) First four histograms as in Figure 1. Histogram 5 shows background fluorescence (FL2) of lymphocytes gated from region E, histogram 1. Histogram 6 shows background fluorescence (FL2) of CD34+ cells gated from region D, histogram 4. (b) Representative fluorescence histograms for the four subsets studied. All histograms gated on CD34+ cells and stained with PE subset antibodies. Cursor position was determined from (a), histogram 6. Histogram 1–4 CD34/38, CD34/CD90, CD34/CD33 and CD34/CD133.

Bone Marrow Transplantation Filgrastim and ancestim in patients with MM IH Chin-Yee et al 856 Table 2 CD34+ cell enumerator from leukapheresis samples that the mobilization regimen combined with large-volume leukapheresis was a critical factor in harvesting such large WBC CD34% Day 11 Days 11 + 12 numbers of progenitor cells. In a randomized study of ϫ109/l CD34/kg CD34/kg patients with multiple myeloma with similar prior chemotherapy, Facon et al41 showed an increased median yield = Non-heavily pretreated (n 57) of CD34+ cells and a reduced number of leukapheresis pro- Average 476.1 3.3 49.4 51.1 Median 432.8 2.9 37.9 39.5 cedures in patients receiving ancestim, filgrastim, and Minimum 36.5 0.1 1.9 5.2 cyclophosphamide compared to filgrastim and cyclophos- Maximum 1250 11 221.3 221.3 phamide alone. Enhanced CD34+ mobilization when fil- Heavily pretreated (n = 8) grastim is combined with ancestim, compared to filgrastim Average 316.5 0.9 5.3 8.0 alone, has been observed in a number of other studies in Median 300.5 0.5 3.3 5.7 breast cancer,32,39 non-Hodgkin’s lymphoma,42,43 and Minimum 114 0.1 1.3 2.8 47 + Maximum 705 3.9 25.3 25.3 ovarian cancer. Higher yields of CD34 cells are associated with more rapid hematopoietic recovery in PBSC transplantation,46,47 but combination cytokine therapy including ancestim has yet to demonstrate a significant clinical impact 32,40,41 blood CD34+ counts was day 11 (median, 665 ϫ 106/l, on engraftment kinetics. For neutrophil engraftment, range, 76 to 1481 ϫ 106/l) (Table 3). the kinetics of recovery are unlikely to be affected significantly once a CD34+ cell dose exceeds 5 ϫ 106/kg.46 Others Subset analysis was also performed on the first cohort of + + Ϫ Ϫ + ϫ 6 20 patients. CD34 subsets CD38 , CD33 , and CD133 have noted that a CD34 cell dose up to 10 10 /kg has all peaked at day 11. CD34+ subset CD90+ peaked at day been associated with progressively more rapid platelet 16 10 with a slight decrease on day 11 (Table 4). recovery. The combination of r-metHuGCSF/r-metHuSCF and cyclophosphamide was well tolerated in the majority of Discussion patients. One allergic reaction characterized by wheezing and shortness of breath after r-metHuSCF was noted in our In our study, 96.9% of evaluable patients (100% in non- study and has been reported by others.43 This was not pre- heavily pretreated, 75% in heavily pretreated) achieved the vented by premedication with ranitidine, cetrizine and sal- target yield of у5ϫ 106 CD34+ cells/kg and 98% achieved butamol. Allergic reaction to SCF is postulated to be caused a ‘minimum target’ of CD34+ of 2 ϫ 106/kg in a one or by mast cell stimulation44,45 and administration of SCF two large-volume leukaphereses. These results represent a should be done in a supervised setting. greater proportion of patients than reported in trials using One potential application of combination cytokine regi- other cytokines with or without chemotherapy mobilization mens, such as ancestim and filgrastim, may be enhanced strategies. In most of these studies, the median number of mobilization in heavily pretreated patients who might other- leukaphereses was two or more.38–40 Although the overall wise not be considered candidates for transplantation patient population in this study was not heavily pretreated, because of excessive prior alkylator exposure. Tricot et al8 the median absolute yield of CD34+ cells obtained suggests found in patients with multiple myeloma that a CD34+

Table 3 CD34+ cell enumeration of peripheral blood and leukapheresis samples (n = 20)

Day 0 Day 9 Day 10 Day 11 Day 12

Peripheral blood samples LKC (ϫ109/l) Median 5.8 12.8 38.7 61.4 61 Range 3.1–10.1 1.5–50.0 12.2–81.0 35.2–103.0 37.9–95.8 CD34 (ϫ 106/l) Median 2 281 414 665 297 Range 0–634–750 63–1095 76–1481 25–822

Leukapheresis samples LKC (ϫ109/l) Median 505.2 Range 232.8–1250 CD34 (ϫ 106/PACK)a Median 4087 Range 538–11815 CD34 (ϫ106/kg) Median 48.8 Range 9.1–221.2

LKC = leukocyte count. aTotal CD34 collected/per apheresis.

Bone Marrow Transplantation Filgrastim and ancestim in patients with MM IH Chin-Yee et al 857 Table 4 Subset analysis of CD34+ cells from peripheral blood and leukapheresis samples (n = 20)

Day 9 Day 10 Day 11 Day 12

Peripheral blood samples CD34+CD38Ϫ (ϫ106/l) Median 0.41 0.66 3.22 2.39 Range 0–4.39 0–7.72 0–11.60 0.25–15.15 CD34+CD90+ (ϫ106/l) Median 130.6 147.0 120.0 43.9 Range 17.7–231.7 21.9–336.3 22.0–360.7 8.53–206.3 CD34+CD33Ϫ (ϫ106/l) Median 15.1 37.9 119.1 55.0 Range 0.6–243.1 2.86–525.6 9.8–753.2 4.8–702.8 CD34+AC133+ (ϫ106/l) Median 226.4 371.8 509.8 208.7 Range 21.7–610.5 44.5–823.4 55.6–1134.5 14.9–568.8

Leukapheresis samples CD34+CD38Ϫ (ϫ106/l) Median 0.2 Range 0.0–1.22 CD34+CD90+ (ϫ106/l) Median 5.17 Range 1.10–47.40 CD34+CD33Ϫ (ϫ106/l) Median 10.8 Range 0.98–90.0 CD34+AC133+ (ϫ106/l) Median 33.2 Range 5.8–150.5

threshold of 2 ϫ 106/kg was obtained in 97% of patients patients who had peripheral blood CD34+ counts monitored with shorter exposure (12 months of prior chemotherapy), on day 11, it is possible that 69% could have obtained a 85% of patients with 13 to 24 months of exposure, and minimum CD34+ cell count of 2 ϫ 106/kg by donating 500 28% of patients with greater than 24 months of prior ther- ml of whole blood and 82% of patients would have apy. Although the number of heavily pretreated patients in obtained the minimum CD34+ cell number (2 ϫ 106/kg) this study, defined as having received greater than six after donation of 1 liter of whole blood. The feasibility of cycles of chemotherapy or more than two cycles containing whole blood donation to engraft patients after myeloabl- melphalan or nitrosureas (BCNU), was limited, many of ative therapy has previously been demonstrated.50,51 Whole these patients had been exposed to multiple cycles of blood donation as a source of PBSC has the potential to alkylator therapy (median, 5; range: 3–37). Of the five entirely eliminate the requirement for leukapheresis and patients who had received more than 24 months of chemo- avoid the loss of stem cells associated with cryopreserv- therapy, three (60%) achieved the target yield and the ation.52,53 remaining two (40%) achieved the minimum of 2 ϫ In addition to measuring the absolute yield of CD34+ 106CD34+ cells/kg. One of those patients had 18 cycles of cells, we monitored the kinetics of subsets of CD34+ cells melphalan and had failed filgrastim mobilization on two mobilized using this regimen. We observed a temporal previous occasions. Although showing delayed release of increase in all subsets from day 9 to day 11, with the excep- CD34+ cells into the peripheral circulation, 2.3 ϫ 106 tion of CD90. These results are consistent with previous CD34+ cells/kg in three leukapheresis procedures were col- studies examining the mobilization kinetics of CD90 (Thy- lected and the patient proceeded to infusion and 1).11,54,55 Previous studies14,16 have shown that the speed of engraftment. Thus, although heavily pretreated patients had engraftment relates to the number of CD34+ CD33Ϫ cells lower yields, a proportion of these patients can mobilize infused. Specifically, Pecora et al14 suggested that even adequate grafts and should not necessarily be excluded among patients who received Ͼ 5 ϫ 106 CD34+ cells/kg, from PBSC transplantation. those who received Ͻ1 ϫ 106 CD34+ CD33Ϫ cells demon- Predictable timing and a reduced requirement for leu- strated poor engraftment compared to patients who received kapheresis in patients receiving ancestim and filgrastim Ͼ 1ϫ 106 CD34+ CD33Ϫ cells. In our analysis, all patients may be advantageous with respect to the cost and morbidity had at least 1 ϫ 106 CD34+ CD33Ϫ cells in their leukapher- associated with multiple leukapheresis procedures.49 Since esis collection. The absolute number of CD133+ yields a the absolute number of CD34+ cells in mobilized peripheral similar correlation to engraftment kinetics as CD34+ cells.56 blood was increased with the combination of ancestim and The CD34+ CD133+ subset, however, contains the majority filgrastim after chemotherapy, it raises the possibility of of CD34+ cells, but does not contain late pre-B cells or successfully collecting an adequate number of PBSC by some late-committed myeloid progenitor cells.57 For whole blood donation.41 Of the nonheavily pretreated patients heavily pretreated who may produce low total

Bone Marrow Transplantation Filgrastim and ancestim in patients with MM IH Chin-Yee et al 858 numbers of CD34+ cells and in which a large proportion 4 Fermand JP, Ravaud P, Katsahian S et al. High dose therapy may be pre-B cells, this antibody may prove useful in (HDT) and autologous blood stem cell (ABSC) transplantation assessing graft adequacy. versus conventional treatment in multiple myeloma (MM): CD34+ CD38Ϫ progenitors are thought to contain the results of a randomized trial in 190 patients 55 to 65 years of most primitive stem cells capable of repopulating age. Blood 1999; 94 (Suppl. 1): 396a (Abstr.). 5 Lenhoff S, Hjorth M, Holmberg E et al. Impact on survival of NOD/SCID mice. Some studies have suggested a corre- + Ϫ high-dose therapy with autolotous stem cell support in patients lation with the number of CD34 CD38 cells infused and younger than 60 years with newly diagnosed multiple myel- 13,17 engraftment. Technically, this fraction is the most dif- oma: a population-based study. Blood 2000; 95:7–11. + Ϫ ficult to enumerate as, in most patients, the CD34 CD38 6 Henry JM, Sykes PJ, Brisco MJ et al. Comparison of myeloma cells represent Ͻ 2% of all CD34+ cells. Nevertheless, by cell contamination of bone marrow and peripheral blood stem counting a minimum of 1000 CD34+ events and by using cell harvests. Br J Haematol 1996; 92: 614–619. stringent gating criteria, it was possible to identify a small 7 Harousseau JL, Attal M, Divine M et al. Comparison of auto- population of CD34+ CD38Ϫ cells in all patients in our logous bone marrow transplantation and peripheral blood stem study. A significant yield of CD34+ CD38Ϫ cells using a cell transplantation after first remission induction treatment in similar mobilization regimen was also reported by Weaver multiple myeloma. Bone Marrow Transplant 1995; 15: 963– 31 969. et al. 8 Tricot G, Jagannath S, Vesole D et al. Peripheral blood stem Human and animal studies have suggested that the + + cell transplants for multiple myeloma: identification of favor- CD34 CD90 fraction of cells in PBSC contain all of the able variables for rapid engraftment in 225 patients. Blood cells with engraftment potential.11,55 Three studies have 1995; 85: 588–596. shown that small numbers of highly selected cell popu- 9 Hohaus S, Goldschmidt H, Ehrhardt R, Haas R. Successful lations for CD34+ CD90+ linϪ were capable of engrafting autografting following myeloablative therapy with blood stem patients.15,58,59 Negrin et al17 found rapid engraftment in all cells mobilized by chemotherapy plus rhG-CSF. Exp Hematol breast cancer patients given CD34+ CD90+ linϪ cells at a 1993; 21: 508–514. dose of Ͼ8 ϫ 105/kg. In our study, this particular popu- 10 Schwartzberg L, Birch R, Blanco R et al. Rapid and sustained lation of CD34+ CD90+ cells peaked earliest during mobil- hematopoietic reconstitution by peripheral blood stem cell infusion alone following high-dose chemotherapy. Bone Mar- ization with the greatest proportion (median, 50%) on day row Transplant 1993; 11: 369–374. 9 and the highest absolute number on day 10 as opposed 11 Prince HM, Imrie K, Sutherland DR et al. Peripheral blood to day 11 for the other subsets analyzed. Previous studies progenitor cell collections in multiple myeloma: predictors have also demonstrated early mobilization kinetics for this and management of inadequate collections. Br J Haematol subset using different mobilization regimens.11 Although 1996; 93: 142–145. early mobilization could have implications for positive 12 Bensinger WI, Longin K, Appelbaum F et al. 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