CD34 -selected autologous peripheral blood stem cell ... - Nature

Bone Marrow Transplantation, (1997) 20, 827–834  1997 Stockton Press All rights reserved 0268–3369/97 $12.00

CD341-selected autologous peripheral blood stem cell transplantation (PBSCT) in patients with poor-risk hematological malignancies and solid tumors. A single-centre experience D Nachbaur1, F-M Fink2, W Nussbaumer3, A Ga¨chter1, G Kropshofer1, C Ludescher4 and D Niederwieser1 1

Division of Clinical Immunobiology and Bone Marrow Transplantation, Department of Internal Medicine, 2Department of Pediatrics, and 3 Department of Transfusion Medicine, 4Department of Hematology-Oncology, University Hospital, Innsbruck, Austria

Summary: Between July 1994 and December 1996, PBSC were mobilized in 28 patients with poor-risk hematological malignancies and solid tumors. CD341 cells were positively immunoselected using the Ceprate CS System. By December 1996, 22 patients had been reinfused with a median of 3.325 (0.078–9.5) 3 106/kg CD341 cells. In three patients unselected back-up PBSC had to be transfused along with selected CD341 cells because of a CD341 cell number ,0.5 3 106/kg. G-CSF (10 mg/kg) was started on day 11 and all patients engrafted within a median day number of 12 (range, 10–22) until leukocytes .1.0 3 109/l and a median day number of 56 (range, 10–180) until platelets .20.0 3 109/l (ie platelet transfusion independence). Time to leukocyte and platelet recovery was significantly shorter in patients receiving .2.0 3 106/kg purified CD341 cells as compared to patients reinfused with ,2.0 3 106/kg CD341 cells. The hematopoietic recovery time was similar to that of 18 historical control patients treated with unseparated ABMT 6 PBSCT with the exception of a significantly faster leukocyte engraftment in patients receiving .2.0 3 106/kg CD341 cells and a significantly delayed platelet recovery time in patients receiving ,2.0 3 106/kg purified CD341 cells. There was a trend for a better overall survival and a lower probability of progression/relapse as compared to the historical controls. We observed five episodes of serious opportunistic infections (three pulmonary fungal infections, two cases of cryptosporidiosis) after the take. Four of these patients had been reinfused with ,2.0 3 106/kg CD341 cells probably indicating a delayed immune reconstitution after CD341 -selected PBSCT. Keywords: CD34+ selection; peripheral blood stem cell transplantation; hematological malignancies; solid tumors

Correspondence: Dr D Nachbaur, Division of Clinical Immunobiology and Bone Marrow Transplantation, Department of Internal Medicine, Anichstrasse 35, A-6020 Innsbruck, Austria Received 11 April 1997; accepted 13 July 1997

Peripheral blood stem cells (PBSC) are increasingly being used for treatment of hematological malignancies and solid tumors.1–8 Incomplete tumor eradication or malignant cells contaminating the autograft contribute to treatment failure in up to 40–60% of patients after ABMT/PBSCT.9–13 Positive selection of CD34+ cells as one approach to ameliorate these shortcomings results in a 1–4 log 10 reduction of tumor cells in apheresis products, but whether this cost- and timeconsuming technique improves survival is currently under investigation.14–16 We report here on our experience in 28 patients suffering from poor-risk malignancies, for whom CD34+-selected PBSC were collected between July 1994 and December 1996. By December 1996, 22 patients had received an autograft with CD34+-selected PBSC. Stem cell collection data are presented and clinical outcome is compared to a historical control group autografted with unselected BM 6 PB stem cells.

Patients and methods Patients, cytapheresis and cryopreservation Between July 1994 and December 1996, stem cells were mobilized in 28 patients, 1–59 years old (median, 39 years) with poor-risk hematological malignancies or solid tumors with high-dose CY (7 g/m2, n = 19) or other dose-intensive chemotherapy regimens (n = 6) and rhG-CSF (10 mg/kg). By December 1996, 22 patients had been autografted with purified CD34+ cells. The characteristics of these 22 patients are listed in Table 1. All patients were considered to have poor-risk disease according to one of the following criteria: multiple lines of treatment prior to transplantation, refractory or progressive disease implying ,50% tumor reduction from initial treatment or .25% increase in tumor size, intermediate- or high-grade lymphoma achieving only partial remission after standard chemotherapy, solid tumors with metastatic disease at initial diagnosis or local or disseminated relapses, stage IIb to IV or inflammatory breast cancer. PBMC were collected in the recovery phase, when CD34+ cells in the peripheral blood reached values .0.3% using a CS 3000 Plus blood cell separator (Baxter Healthcare, Fenwal Division, Deerfield, IL, USA). Three leukaphereses were performed for each individual patient.

M M M F M M F F M M F M F M M F M M M F F M

153 157 161 163 164 171 175 177 181 186 190 192 196 197 200 201 202 203 204 210 213 216

3 55 42 45 52 51 1 22 3 42 33 33 15 36 2 42 4 59 51 50 51 23

Age (years)

Neuroblastoma Multiple myeloma Multiple myeloma Multiple myeloma Multiple myeloma High-grade NHL (Ki1+) Wilm’s tumour Hodgkin’s disease Rhabdomyosarcoma High-grade NHL (Ki1+) Breast cancer Nonseminomatous germ cell tumor Neuroblastoma Hodgkin’s disease Neuroblastoma Intermediate-grade NHL Rhabdomyosarcoma Multiple myeloma Multiple myeloma Breast cancer Follicular NHL, Hodgkin’s disease Ewing sarcoma

Diagnosis

IV III A III A III A III A IV B IV 3rd relapse after ABMT IV IV IIb metastatic disease IV II B, bulky disease IV IV B IV III A III A IIb (bilateral) IVB metastatic disease

Stage at diagnosis

CR PR SD refractory PCL refractory CR CR CR refractory CR PD PR PD very good PR PR CR PD refractory CR CR CR

Stage at TX

A-NB-94 (6 cycles) VAD, RT MP (.3a), IFN VAD MP, RT, VAD COP, CHOP, IMVP-16 SIOP-93-01, HD-CAV COPP/ABVD, ABMT, RT CWS-91 (8 cycles), RT COP, CHOP, IFN EF PEB, PEI A-NB 94 (6 cycles) RT, COPP/ABVD, IEV A-NB 94 (8 cycles) COP, CHOP CWS-96 (9 cycles) MP, VAD, RT, IFN MP, VAD CMF COPP/ABVD, COP, NOSTE, IFN VAIA, CP/VP-16/CY, RT

Previous treatment

7 9 62 17 29 63 21 54 7 19 12 64 6 25 7 15 7 43 42 6 101 108

Interval DX to TX (months)

CR CR CR PR PD CR relapse CR relapse PD CR PD CR SD CR CR CR PR SD CR CR CR

Response to TX

NHL = non-Hodgkin’s lymphoma; AILD = angioimmunoblastic T cell lymphoma; PCL = plasma cell leukemia; ABMT = autologous bone marrow transplantation; CR = complete remission; PR = partial remission; SD = stable disease; A-NB-94 = Austrian Neuroblastoma Study, protocol 94; VAD = vincristine, doxorubicin, dexamethasone; RT = radiotherapy; MP = melphalan, prednisone; IFN/P = interferon/prednisone; COP = cyclophosphamide, vincristine, prednisone; CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone; IMVP-16 = ifosfamide, methotrexate, etoposide; SIOP93-01 = International Society of Pediatric Oncology, protocol 93-01; HD-CAV = high-dose cyclophosphamide, doxorubicin, vincristine; COPP/ABVD, cyclophosphamide, vincristine, procarbazine, prednisone/doxorubicin, bleomycin, vinblastine, DTIC; CWS-91 = Cooperative Study on Soft Tissue Sarcomas, protocol 91; EC = epirubicin, cyclophosphamide; IEV = ifosfamide, epirubicin, etoposide; CMF = cyclophosphamide, methotrexate, 5-FU; NOSTE = mitoxantrone, prednimustine; VAIA = ifosfamide, doxorubicin, dactinomycin, vincristine; CP = carboplatin; VP-16 = etoposide; CY = cyclophosphamide.

Sex

Characteristics of 22 patients autografted with CD34+-selected PBSC

UPN

Table 1

CD341-selected autologous PBSCT D Nachbauer et al

828


The first two apheresis products were pooled and CD34+ cells were positively immunoselected with a CD34-specific, biotinylated MoAb (12–8) according to the manufacturer’s instructions using the Ceprate SC Stem Cell Concentration System (CellPro, Bothell, WA, USA). Cells were cryopreserved in 10% DMSO by control-rated freezing and stored in liquid nitrogen. The third apheresis product was cryopreserved unselected as back-up in all patients. The historical control group consisted of 18 consecutive patients with poor-risk malignancies according to the above-mentioned criteria autografted at our institution between 1983 and 1996 (solid tumor, n = 11; lymphoma, n = 3; multiple myeloma, n = 4). The two groups were not different with respect to diagnosis, median age at transplant, median time to transplantation, lines of previous treatment, and median post-transplant follow-up (P = NS). There were significantly more females in the historical control group (11/18 vs 8/22, P = 0.0027) and significantly more patients in the control group received a TBI-containing conditioning regimen (10/18 vs 4/22 in the CD34+ group, P = 0.0001). Patients received unselected hematopoietic stem cells from autologous BM plus PB (n = 11) or PB alone (n = 7). Patients who received BM plus PB stem cells were reinfused with a median of 0.25 (range, 0.1– 0.37) 3 108/kg BM plus 0.95 (range, 0.18–4.34) 3 108/kg PB mononuclear cells. Patients who were autografted with PBSC alone received a median of 4.43 (range, 2.33– 7.66) 3 108/kg PB mononuclear cells. Twelve patients received G-CSF (10 mg/kg/day) beginning on day 1 posttransplant to accelerate hematopoietic reconstitution (vs all patients in the CD34+ group, P = 0.0002).

CD34 quantitation Cell counts were performed using a fluorescence-activated cell sorter (FACS Vantage; Becton Dickinson, Palo Alto, CA, USA). CD34+ cells were determined daily after stem cell mobilization with high-dose chemotherapy in heparinized venous blood samples when leukocytes reached values .1.0 3 109/l, and in the leukapheresis products, the flow-through fraction from the CEPRATE column, and in the adsorbed fraction using a phycoerythrin (PE)-conjugated anti-human CD34 MoAb (HPCA-2; Becton Dickinson, Palo Alto, CA, USA). Peripheral blood mononuclear cells (PBMC) from heparinized venous blood samples were obtained by Ficoll–Isopaque (Lymphoprep; Nycomed, Oslo, Norway) gradient centrifugation (400 g for 30 s at room temperature). After two washes cells were resuspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin and sodium azide and simultaneously stained with anti-CD34 MoAb and fluorescein (FITC)-conjugated anti-CD45 MoAb (HLe-1; Becton Dickinson) for 20 min at 4°C. Isotype-matched control antibodies were processed in parallel. After two washes, the percentage of CD34+ cells was determined by gating on forward (FSC) and side scatter (SSC) characteristics and CD45 staining. A minimum of 30 000 events were collected in list mode and processed with a CELLQuest software. MNC from the leukapheresis products, the flow-through fraction from the CEPRATE column and from the adsorbed

fraction were stained without Ficoll–Isopaque centrifugation. Transplant procedure, supportive care and maintenance therapy Patients were treated under strict reverse isolation without laminar air-flow. No prophylactic systemic antibiotics were administered. All patients underwent a non-absorbable oral gut decontamination with vancomycin, gentamycin and nystatin. Pneumocystis carinii prophylaxis was performed with trimethoprim-sulfamethoxazole given in a 10-day course before transplantation and after the take. Cytomegalovirus (CMV) pneumonia prophylaxis consisted of infusions of CMV hyperimmunoglobulin (1 ml/kg) every other week until day +100. Irradiated (25 Gy), leukocytedepleted red cell and platelet transfusions from single donors were administered when hemoglobin levels were 7.0 g/dl or less and platelets were 20 3 109/l or less. Pretransplant conditioning regimens consisted of CY (120 mg/kg) and fractionated TBI (12 Gy) in two myeloma patients; busulphan (16 mg/kg) and CY (120 mg/kg) in four myeloma patients; carboplatin (1500 mg/m2), etoposide (60 mg/kg) and melphalan (160 mg/m2) in seven patients with solid tumors; CY (100 mg/kg), BCNU (15 mg/kg) and etoposide (60 mg/kg) in four lymphoma patients; CY (100 mg/kg), etoposide (30 mg/kg) and fractionated TBI (12 Gy) in two lymphoma patients; CY (120 mg/kg), carboplatin (2000 mg/m2) and etoposide (1500 mg/m2) in one patient with nonseminomatous germ cell tumor and carboplatin (800 mg/m2), thiotepa (500 mg/m2), melphalan (90 mg/m2) and novantrone (30 mg/m2) in two breast cancer patients. Forty-eight hours after completing pretransplant conditioning, purified CD34+ cells were rapidly thawed and given intravenously. All patients received G-CSF (10 mg/kg) starting on day +1 given as a 2-h infusion until leukocyte engraftment, defined as the first of 2 consecutive days with leukocyte counts .1.0 3 109/l (take). In three patients additional unselected back-up PBMC were transfused because of the low number of CD34+ cells (,0.5 3 106/kg). Patients with multiple myeloma received recombinant interferon-a-2b (Intron A; Schering International, Vienna, Austria) subcutaneously at a dosage of 2 mega units three times weekly starting after stable engraftment, defined as leukocyte counts .3 3 109/l and platelets .45 3 109/l. Interferon treatment was maintained until disease progression. Patients with radiosensitive bulky or metastatic disease at the time of autografting received involved field irradiation within the first 3 months. Response criteria In patients with multiple myeloma, complete remission (CR) was defined as less than 5% bone marrow plasma cells, no measurable paraprotein in serum and/or urine by immunoelectrophoresis or immunofixation, and no evidence of progressive bony lesions. Partial response (PR) was defined as >75% reduction of bone marrow plasmacytosis and/or paraprotein levels without progressive bony lesions. Stable disease (SD) was defined as 25% increase in marrow plasmacytosis and/or paraprotein levels. 4 For patients with lymphoma or solid tumors CR was defined as the disappearance of all clinical and radiographic evidence of disease. PR was defined as reduction of measurable disease by more than 50% without the appearance of new lesions, SD was defined as less than 50% tumor reduction and PD as the appearance of any new tumor lesion and/or increase of measurable disease by more than 25%. Statistics For the comparison of the distribution between the study and control group we used the x2 test for categorical variables and the Wilcoxon test for continuous variables. Unadjusted Kaplan–Meier estimates were used to display the incidences of overall survival, progression-free survival and transplant-related mortality.17 Progression-free and overall survival were defined as the time from the day of stem cell transplantation (day 0) until disease progression or death. Correlations between CD34+ cell number and time until take or platelet transfusion independence were computed using the Spearman rank correlation coefficient.18 For comparisons of time to engraftment between the independent groups we used the Wilcoxon rank-sum test.19 A P value 1.0 g/l

22

20

18

16

14

12

10

8 0

1

2

3

4

5

6

7

8

9

10

CD34+ cell number (×106/kg)

Discussion b 200

r = –0.7 P = 0.0011

180

Days until platelets >20.0 g/l

160 140 120 100 80 60 40 20 0 0

1

2

3

4

5

6

7

8

9

10

CD34+ cell number (×106/kg) Figure 1 Correlation between the number of reinfused CD34+ cells/kg and time to leukocyte engraftment (a) or platelet transfusion independence (b). The lines represent median values of reinfused CD34+ cell number and of days until leukocyte recovery or platelet transfusion independence. Correlations were computed using the Spearman correlation coefficient.

Autologous transplantation with mobilized PBSC has been shown to be superior to BM stem cells with respect to engraftment kinetics but without any significant differences in relapse incidence, transplant-related mortality or survival.1,3,8,20 Interestingly, in one recent study an even higher relapse incidence after unpurged PBSCT for lymphoma was reported as compared to antibody-purged ABMT.1 Concomitant tumor cell mobilization by the stem cell mobilization procedure has been noticed and tumor cells contaminating autografts have been identified by gene-marking studies as a cause of relapse after ABMT/PBSCT.9–13 Positive selection of CD34+ cells is one possibility to reduce the quantity of contaminating tumor cells. However, the questions to be addressed with this technique include the following: is it possible to mobilize a sufficient number of CD34+ cells even in extensively pretreated, poor-risk patients, do CD34+-selected stem cells mediate a faster hematopoietic regeneration than unseparated stem cells, and is there any advantage with respect to overall survival, transplant-related mortality or relapse incidence after CD34+-selected PBSCT. Using the Ceprate SC Stem Cell Concentration System we obtained a sufficient number of CD34+ stem cells even in intensively pretreated poor-risk patients. All but one of nine patients in which ,2.0 3 106/kg CD34+ cells were


a

Overall survival (% probability)

100

80

57%, CD34+ selected (n = 22)

60

40

16%, unselected (n = 18) 20

0 0

1

2

3

4

5

6

7

8

9

10

11

Years

Transplant-related mortality (% probability)

b 40

30

18%, unselected (n = 18) 20

10

5%, CD34+ selected (n = 22) 0 0

0

2

3

4

5

6

7

8

9 10 11

Years

c 100

Relapse/progression (% probability)

832

75%, unselected (n = 18)

80

60

40

25%, CD34+ selected (n = 22)

20

0 0

1

2

3

4

5

6

7

8

9

10

11

Years Figure 2 Probability of overall survival (a), transplant-related mortality (b) and relapse/progression (c) after CD34+-selected PBSCT (n = 22) and unselected ABMT 6 PBSCT (n = 18).

obtained had received >3 lines of previous treatment including either alkylating agents or irradiation or had bone marrow involvement of their underlying disease, factors that have all been identified as significant determinants of progenitor cell yield.2,20,21 Additionally, three of these nine patients also experienced prolonged treatment with IFN-a prior to stem cell collection. Suppression of myeloid progenitors by interferon treatment has been demonstrated and must be taken into account as an additional factor adversely affecting stem cell harvests.22 The rates of neutrophil and platelet recovery of the entire CD34+-selected group were equivalent when compared with those of the historical control group. These results confirm the observations made in two other small series using positively selected CD34+ cells for hematopoietic reconstitution after high-dose chemotherapy.14,23 Our observation of a significantly delayed platelet recovery in patients autografted with ,2.0 3 106/kg purified CD34+ cells as compared to those receiving either a higher cell dose of selected CD34+ or unselected BM 6 PB stem cells is in line with the recently published data by Weaver et al24 about platelet reconstitution kinetics in patients autografted with unselected PBSC containing a low CD34+ cell dose and suggests that G-CSF alone is not sufficient to induce a parallel increase of both granulocytes and platelets in this group of patients.24 Whether the use of post-transplant GCSF can even delay platelet recovery in patients receiving a low CD34+ cell dose as it was demonstrated in a large retrospective analysis by the Seattle group remains to be shown in a prospective randomized trial.2 The third question of whether there is any survival advantage for patients autografted with purified CD34+ cells due to the tumor cell purging effect will not be answered until controlled prospective randomized trials have been completed. However, there was a trend towards a better overall survival due to a lower relapse/progression probability after CD34+-selected PBSCT. Whether ex vivo expansion of purified CD34+ cells resulting in reduction of contaminating tumor cells or combined positive and negative selection procedures could further improve the results is under investigation.25–27 Furthermore, the stem cell mobilization procedure itself using high-dose CY might be a good prognostic parameter and might have contributed to the beneficial effects of CD34+ selection on overall survival and relapse incidence in our poor-risk patients. Another important aspect of CD34+ selection deals with the concomitant T cell depletion and its possible consequences on immune reconstitution kinetics after CD34+selected PBSCT, although purified, allogeneic CD34+ cells devoid of mature T and B lymphocytes have been shown to restore lymphopoiesis in lethally irradiated non-human primates.28 Whether lymphocyte reconstitution after CD34+-selected PBSCT is delayed or even is a function of CD34+ cell number resulting in a higher rate of infectious complications, as it is observed after T cell depletion in the allogeneic setting, is unknown.29 Preliminary observations at our institution reveal a profound delay of qualitative and quantitative T lymphocyte reconstitution, especially of CD4+ cells, within the first 100 days after CD34+-selected PBSCT probably rendering those patients who receive a low CD34+ cell dose (,2.0 3 106/kg CD34+ cells) more


susceptible to opportunistic infections even after successful and prompt leukocyte engraftment.30 In conclusion, CD34+ selection is feasible even in extensively pretreated poor-risk patients. Hematopoietic regeneration was comparable to that of patients receiving unmanipulated BM and/or PB stem cells but, despite prompt neutrophil regeneration, platelet recovery was significantly delayed in patients receiving ,2.0 3 106/kg CD34+ cells. The survival data are encouraging but a prolonged followup and future, randomized studies are necessary to assess the effects of CD34+ selection on survival, relapse incidence and immune reconstitution.

11

12 13

14

Acknowledgements This work was financially supported by grants of the Austrian Research Fund ‘Zur Fo¨rderung der wissenschaftlichen Forschung’, project No. 10525.

15

16

References 1 Brunvand MW, Bensinger WI, Soll E et al. High-dose fractionated total-body irradiation, etoposide and cyclophosphamide for treatment of malignant lymphoma: comparison of autologous bone marrow and peripheral blood stem cells. Bone Marrow Transplant 1996; 18: 131–141. 2 Bensinger W, Appelbaum F, Rowley S et al. Factors that influence collection and engraftment of autologous peripheralblood stem cells. J Clin Oncol 1995; 13: 2547–2555. 3 Langenmayer I, Weaver C, Buckner CD et al. Engraftment of patients with lymphoid malignancies transplanted with autologous bone marrow, peripheral blood stem cells or both. Bone Marrow Transplant 1995; 15: 241–246. 4 Bensinger WI, Rowley SD, Demirer T et al. High-dose therapy followed by autologous hematopoietic stem-cell infusion for patients with multiple myeloma. J Clin Oncol 1996; 14: 1447–1456. 5 Weaver CH, Petersen FB, Appelbaum FR et al. High-dose fractionated total-body irradiation, etoposide, and cyclophosphamide followed by autologous stem-cell support in patients with malignant lymphoma. J Clin Oncol 1994; 12: 2559– 2566. 6 Elias AD, Ayash L, Anderson KC et al. Mobilization of peripheral blood progenitor cells by chemotherapy and granulocyte–macrophage colony-stimulating factor for hematologic support after high-dose intensification for breast cancer. Blood 1993; 79: 3036–3044. 7 Fukuda M, Kojima S, Matsumoto K, Matsuyama T. Autotransplantation of peripheral blood stem cells mobilized by chemotherapy and recombinant human granulocyte colony-stimulating factor in childhood neuroblastoma. Br J Haematol 1992; 80: 327–331. 8 To LB, Roberts MM, Haylock DN et al. Comparison of haematological recovery times and supportive care requirements of autologous peripheral blood stem cell transplants, autologous bone marrow transplants and allogeneic bone marrow transplants. Bone Marrow Transplant 1991; 9: 277–284. 9 Deisseroth AB, Zu Z, Claxton D et al. Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML. Blood 1994; 83: 3068–3076. 10 Brenner MK. Rill DR, Moen RC et al. Gene-marking to trace

17 18 19 20

21

22

23

24

25

26

27

origin of relapse after autologous bone marrow transplantation. Lancet 1993; 341: 85–86. Rill DR, Santana VM, Roberts WM et al. Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 1994; 84: 380–383. Brugger W, Bross KJ, Glatt M et al. Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors. Blood 1994; 83: 636–640. Ross AA, Cooper BW, Lazarus HM et al. Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques. Blood 1993; 82: 2605–2610. Lemoli RM, Fortuna A, Motta MR et al. Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells. Blood 1996; 87: 1625–1634. Schiller G, Vescio R, Freytes C et al. Transplantation of CD34+ peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. Blood 1995; 86: 390–397. Shpall EJ, Jones RB, Bearman SI et al. Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: influence of CD34-positive peripheral-blood progenitors and growth factors on engraftment. J Clin Oncol 1994; 12: 28–36. Kaplan E, Meier P. Non-parametric estimation for incomplete observations. J Am Stat Assoc 1958; 53: 457–481. Brown BW. Hollander M. Linear regression and correlation. In: Brown B, Hollander M (eds). Statistics. Wiley: New York, 1977, pp 261–307. Sachs L. Angewandte Statistik. Springer Verlag: Berlin, 1974. Tricot G, Jagannath S, Vesole D et al. Peripheral blood stem cell transplants for multiple myeloma: identification for favorable variables for rapid engraftment in 225 patients. Blood 1995; 85: 588–596. Dreger P, Klo¨ss M, Petersen B et al. Autologous progenitor cell transplantation: prior exposure to stem cell toxic drugs determines the yield and engraftment of peripheral blood progenitor cell but not of bone marrow grafts. Blood 1995; 86: 3970–3978. De Fabritiis P, Sandrelli A, Meloni G et al. Prolonged suppression of myeloid progenitor cell numbers after stopping interferon treatment for CML may necessitate delay in harvesting marrow cells for autografting. Bone Marrow Transplant 1990; 6: 247–251. Brugger W, Henschler R, Heimfeld S et al. Positively selected autologous blood CD34+ cells and unseparated peripheral blood progenitor cells mediate identical hematopoietic engraftment after high-dose VP16, ifosfamide, carboplatin, and epirubicin. Blood 1994; 84: 1421–1426. Weaver CH, Potz J, Redmond J et al. Engraftment and outcomes of patients receiving myeloablative therapy followed by autologous peripheral blood stem cells with a low CD34+ cell content. Bone Marrow Transplant 1997; 19: 1103–1110. Willems P, Croockewitt A, Raymakers R et al. CD34 selections from myeloma peripheral blood cell autografts contain residual tumour cells due to impurity, not to CD34+ myeloma cells. Br J Haematol 1996; 93: 613–622. Widmer L, Pichert G, Jost LM, Stahel RA. Fate of contaminating t(14;18)+ lymphoma cells during ex vivo expansion of CD34-selected hematopoietic progenitor cells. Blood 1996; 88: 3166–3175. Di Nicola M, Bregni M, Siena S et al. Combined negative and positive selection of mobilized CD34+ blood cells. Br J Haematol 1996; 94: 716–721.

833


834

28 Andrews RG, Bryant EM, Bartelmez SH et al. CD34+ marrow cells, devoid of T and B lymphocytes, reconstitute stable lymphopoiesis and myelopoiesis in lethally irradiated allogeneic baboons. Blood 1992; 80: 1693–1701. 29 Pirsch JD, Maki DG. Infectious complication in adults with bone marrow transplantation and T cell depletion of donor marrow. Increased susceptibility to fungal infections. Ann Intern Med 1986; 104: 619–631.

30 Nachbaur D, Kropshofer G, Feichtinger H et al. Cryptosporidiosis after CD34-selected autologous peripheral blood stem cell transplantation (PBSCT). Treatment with paromomycin, azithromycin and recombinant human interleukin-2. Bone Marrow Transplant 1997; 19: 1261–1263.