Predictive factors for long-term engraftment of autologous blood stem cells

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1 (2000) 26, Macmillan Publishers Ltd All rights reserved /00 $ Predictive factors for long-term engraftment of autologous blood stem cells PR Duggan 1, D Guo 2, J Luider 3, I Auer 3, J Klassen 4, A Chaudhry 1, D Morris 1, S Glück 1, CB Brown 1, JA Russell 1 and DA Stewart 1 Departments of 1 Medicine, 2 Epidemiology, 3 Flow Cytometry, and 4 Apheresis, Tom Baker Cancer Center, Foothills Hospital, and University of Calgary, Calgary, Alberta, Canada Summary: Data from 170 consecutive patients aged years (median age 46 years) who underwent unmanipulated autologous blood stem cell transplant (ASCT) were analyzed to determine if total CD34 + cells/kg infused, CD34 + subsets (CD , CD , CD , CD , CD DR ), peripheral blood CD34 + cell (PBCD34 + ) count on first apheresis day, or various clinical factors were associated with low blood counts 6 months post ASCT. Thirty-four patients were excluded from analysis either because of death (n = 17) or reinduction chemotherapy prior to 6 months post ASCT (n = 13), or because of lack of follow-up data (n = 4). Of the remaining 136 patients, 46% had low WBC ( /l), 41% low platelets ( /l), and 34% low hemoglobin ( 120 g/l) at a median of 6 months following ASCT. By Spearman s rank correlation, both the total CD34 + cell dose/kg and the PBCD34 + count correlated with 6 month blood counts better than any subset of CD34 + cells or any clinical factor. The PBCD34 + count was overall a stronger predictor of 6 month blood counts than was the total CD34 + cells/kg infused. Both factors retained their significance in multivariate analysis, controlling for clinical factors. In conclusion, subsets of CD34 + cells and clinical factors are inferior to the total CD34 + cell dose/kg and PBCD34 + count in predicting 6 month blood counts following ASCT. (2000) 26, Keywords: hematopoietic stem cell transplantation; autologous; long-term engraftment; CD34 + subsets The ability of the infused dose of CD34 + cells to predict early hematologic recovery after autologous stem cell transplant (ACST) has been well described. 1 4 More rapid hematopoietic recovery after high-dose chemotherapy (HDCT) and ASCT is in turn associated with fewer red blood cell and platelet transfusions, fewer febrile days, less antibiotic use, shorter hospitalization and lower supportive care costs. 4 6 Subsets of CD34 + cells expressing markers of lin- Correspondence: Dr DA Stewart, Tom Baker Cancer Center, th Street NW, Calgary, Alberta, Canada T2N 4N2 Received 9 June 2000; accepted 12 September 2000 eage commitment can be detected and enumerated using flow cytometric techniques. 7 These committed progenitor cells, expressing myeloid (CD33, CD13), 8 erythroid (CD71), 9 and megakaryocytic (CD41, CD61) 10 markers, have also shown some correlation with engraftment following ASCT. 1,2,4,11 However, controversy exists regarding the relative merit of using the dose of CD34 + subsets for predicting the early reconstitutive capacity of autologous stem cell grafts. 1 There is considerably less information available about possible factors that affect long-term hematopoietic engraftment after ASCT. Lower numbers of CD34 + cells/kg have been reported to correlate with the presence of abnormal peripheral blood counts at both 6 and 12 months post ASCT Patients receiving a larger cell dose may experience less fever and require fewer transfusions, antibiotics, and days in hospital in the first year after transplantation. 13 However more information is needed about the relative influence of CD34 + cell dose, CD34 + subsets, and clinical factors on long-term hematopoiesis. In this retrospective study, we have evaluated the effect of CD34 + cell dose, peripheral blood CD34 + cell count, and CD34 + subsets on long-term peripheral blood counts (WBC, hemoglobin, and platelets) 6 months following ASCT. In addition, we studied the influence of pretransplant clinical factors on these outcomes. Materials and methods Patients One hundred and seventy consecutive patients underwent a single ASCT with unmanipulated autografts in Calgary between January 1997 and November Thirty-four patients were excluded from analysis either because of death (n = 17) or re-induction chemotherapy within 6 months of ASCT (n = 13), or because of lack of follow-up data (n = 4). Patients with evidence of relapsed or persistent disease after autografting were included if no systemic therapy was given before 6-month blood counts could be evaluated. The characteristics of the remaining 136 patients are listed in Table 1. Nineteen patients (14%) had marrow involvement prior to mobilization. A small group of four patients received hematopoietic growth factor-only mobilization with G-CSF daily for at least 3 days and apheresis starting on day 4. A second group

2 1300 Table 1 Pre-transplant patient characteristics Characteristic No. patients % Age (median) years (46 years) Gender M F Diagnosis Breast cancer Non-Hodgkin s lymphoma Hodgkin s lymphoma Multiple myeloma 11 8 Other (medulloblastoma, AML, 10 7 oligodendrioma, amyloidosis, germ cell tumour) Remission status First remission Relapsed Primary refractory 8 6 No. CT regimens Marrow positive pre-transplant Yes No No. aphereses TBI Yes No Consolidative radiotherapy Yes No No. CT = number of prior chemotherapy regimens. of 54 patients received cyclophosphamide 5.25 g/m 2, etoposide 1.05 g/m 2, cisplatin 105 mg/m 2 (DICEP) divided over days 1 3 and G-CSF 300 g ( 70 kg) or 480 g ( 70 kg) s.c. starting on day 14, and were scheduled for apheresis on days 19, 20 or 21. The remaining 78 patients underwent mobilization with G-CSF and single agent or combination chemotherapy dosed to produce short-duration neutropenia and thrombocytopenia that would not require platelet transfusion. These 78 patients received daily G-CSF 300 g ( 70 kg) or 480 g ( 70 kg) s.c. starting on days 7 or 8, and underwent apheresis on days 13 or 14. The most common mobilization regimens for these patients were based upon cyclophosphamide 2 g/m 2 (n = 57), ifosfamide 4.5 g/m 2, etoposide 400 mg/m 2 and cisplatin 100 mg/m 2 (n = 11), taxanes (n = 8) or high-dose Ara-C (n = 2). Blood stem cell apheresis and cryopreservation methods have been previously described. 16 Conditioning regimens for ASCT varied by diagnosis and study protocol. They were all considered to be myeloablative or profoundly myelosuppressive, requiring ASCT for timely blood count recovery. Lymphoma and multiple myeloma patients mainly received melphalan mg/m 2 ± 500 cgy TBI (n = 63). Breast cancer patients received cyclophosphamide 6 g/m 2 and mitoxantrone mg/m 2 plus either vinblastine 12.5 mg/m 2, vinorelbine 85 mg/m 2, or carboplatin 1800 mg/m 2 (n = 57). The other most common regimens were BEAM (BCNU 300 mg/m 2, etoposide 800 mg/m 2, Ara-C 1600 mg/m 2, and melphalan 140 mg/m 2, n = 5) or thiotepa-based (n = 11). Total body irradiation was included in the conditioning regimen for 16% of patients, while 52% received post-transplant consolidation radiotherapy to areas of prior disease involvement. G-CSF at doses of 300 ( 70 kg) or 480 ( 70 kg) g/day s.c. was administered from day +7 post transplant until ANC 1.5. All patients provided written informed consent and all studies were approved by the institutional review board. Blood stem cell enumeration To ascertain the optimal timing for apheresis and ensure the best possible yield of stem cells, CD34 + cell counts were measured in peripheral blood by flow cytometry daily from the fifth day of G-CSF until apheresis was complete. Total CD34 + cell count and CD34 + subsets (CD , CD , CD , CD and CD DR ) were determined from the final apheresis product by flow cytometry and are expressed in terms of the actual count divided by patient weight. Flow cytometry techniques have been previously described. 1 Statistics Measures of long-term marrow engraftment were white blood cell count (WBC), hemoglobin (Hb), and platelet count obtained closest to six months post ASCT. All values were measured at least five months post transplant and ranged from 5 to 24 months (median 6.0 months). Low blood counts in Calgary are defined as WBC /l, Hb 120 g/l and platelets /l. A number of clinical factors that could potentially affect long-term engraftment were analyzed including age, gender, diagnosis, remission status, number of prior chemotherapy regimens, prior radiotherapy, morphological bone marrow involvement, mobilizing regimen, conditioning regimen, use of total body irradiation (TBI) in conditioning, relapse post ASCT, and post-asct consolidation radiotherapy. In univariate analysis, Spearman s rank correlation test was used for continuous independent variables. Kruskal Wallis rank sum test was used for categorical independent variables. Logistic models were then built to study the effect of a predictor with other variables being simultaneously controlled. CD34 + cell count was analyzed as the number of CD34 + cells/kg actually infused. The peripheral blood CD34 + (PBCD34 + ) cell count analyzed was the count measured on the first apheresis day, regardless of how many apheresis procedures a patient underwent. The subsets of CD34 + cells analyzed include CD , CD , CD and CD DR (multipotent progenitors), and CD (megakaryocyte progenitors). Finally, logistic models were used to determine if the various clinical factors, CD34 + cell dose/kg, CD34 + subsets, peripheral blood CD34 + cell count and measures of early engraftment were independently associated with the long-term risk of having low peripheral blood counts after ASCT. The CD34 + cell dose/kg, PBCD34 + cell count, and CD34 + subsets were compared as continuous variables, however, cat-

3 egorical ranges of the CD34 + cell dose/kg and PBCD34 + cell count were used in multivariate analysis. Results A median dose of CD34 + cells/kg was infused (range ). The entire collected product was infused in 107 patients, whereas only half (26 cases) or a third (three cases) of the product was given to 29 patients. The remaining product was stored for possible second transplant for the latter group. None of these 29 patients received the second ASCT before the study endpoint of the 6 month CBC. The median PBCD34 + count on the first apheresis day was /l (range ). A very good correlation was found between (log) PBCD34 + cell count and (log) CD34 + cell dose received (r = 0.86, P ). Engraftment Rapid recovery of marrow function was seen for most patients after ASCT. Patients reached an absolute neutrophil count /l after a median of 11 days (range 8 17 days). A platelet count /l, unsupported by transfusion, was achieved after a median of 11 days (range 8 80 days). Eighty-eight percent were transfusion independent with platelet count /l by day 14 and 98% by day 21. A platelet count /l was achieved by a median of 15 days (range 10 to greater than 185 days), and in 90% by 30 days post ASCT. Five percent of patients never achieved a platelet count /l. In addition, early platelet engraftment did not ensure normal long-term thrombopoiesis as 38% of those patients whose platelets recovered to /l within 30 days of ASCT continued to have thrombocytopenia in the follow-up period, and in 13% the platelet count fell to /l. Long-term engraftment data were available for all 136 patients. Relapse was observed in 15 of them, none of whom had received systemic therapy for relapse prior to being studied. Several months following ASCT, 46% of patients had low WBC, 41% low platelets and 34% low hemoglobin. Abnormalities of only a single lineage were seen in 32% of patients, 27% had low counts in two cell lines, and 11% were pancytopenic with low WBC, hemoglobin and platelets. Normal blood counts were seen in 30%. The median value for WBC was /l (range ), for hemoglobin was 124 g/l (range ) and for platelets /l (range 2 466). Poor engraftment of any one cell line several months after ASCT was significantly associated by univariate analysis with the presence of abnormalities in each of the other two cell lines (association between low WBC and low platelets, P , between low WBC and hemoglobin, P = 0.011, and between low platelets and low hemoglobin, P = 0.005). Predictors of long-term engraftment by univariate analysis By univariate analysis (Spearman s rank correlation, Table 2), all CD34 + subsets studied correlated with low pla- telet counts and the subsets of CD , CD , and CD cells correlated with low WBC several months after ASCT. No individual subset correlated with low hemoglobin. Both the PBCD34 + count and the total CD34 + cell dose/kg infused correlated well with successful long-term engraftment of WBC and platelets, and showed a stronger relationship than any individual subset of CD34 + cells. Table 3 shows the percent of patients with low counts in the long-term after ASCT according to the total CD34 + cell dose received and PBCD34 + count at first apheresis. For the purpose of this analysis, patients were grouped into quartiles based on the CD34 + cell dose 10 6 /kg (1 5, 5 10, and CD34 + cells/kg) and the PBCD34 + count on the first morning of apheresis (5 50, , and /l). In general, the probability of having poor engraftment of a cell line was lowest for those patients who received a CD34+ cell dose /kg or who had a PBCD34 + count /l on the morning of apheresis (Figure 1). Both the PBCD34 + count and the total CD34 + cell dose/kg were predictive of engraftment several months after ASCT (Table 3). The PBCD34 + count was a better predictor of long-term WBC and red cell engraftment while the total CD34 + cell dose/kg was marginally better for long-term platelet engraftment. Several clinical factors had a significant effect on longterm blood counts by univariate analysis. A diagnosis of breast cancer (P = ), female sex (P = ), morphological marrow involvement by disease (P = 0.022), use of non-melphalan containing conditioning regimen (P = 0.002), and conditioning without TBI (P = 0.004) were all associated with a low WBC. Older age ( 46) was associated with low hemoglobin (P = 0.003). Need for more than one apheresis procedure (P = ), TBI containing regimens (P = 0.018), and the number of prior chemotherapy regimens (P = 0.017) were associated with poor long-term platelet engraftment. The use of post-asct consolidation radiotherapy was not associated with long-term engraftment. Predictors of long-term engraftment by multivariate analysis Due to the strong correlation between the PBCD34 + count and the infused CD34 + dose/kg, linear models were set up for each of these two co-dependent variables separately to avoid multicolinearity. When multivariate analysis was applied in this way, and controlling for the various clinical factors, the PBCD34 + count and the total CD34 + cell dose/kg continued to predict for low WBC (P and P , respectively) and platelets (P and P = , respectively). In addition, the PBCD34 + count predicted for anemia when studied in multivariate analysis as a categorical variable (P = 0.037). None of the clinical factors or CD34 + cell subsets showed a consistent relationship with long-term trilineage engraftment by multivariate analysis. Discussion Few studies have addressed the issue of hematopoietic engraftment and marrow function beyond the immediate 1301

4 1302 Table 2 Correlation of CD34 + cell dose, CD34 + subsets, and PBCD34 + count with long-term engraftment post ASCT Subset Median Range Correlation with long-term Correlation with long-term Correlation with long-term WBC Hb platelets rho a P value rho a P value rho a P value Total CD34 + cells 10 6 /kg CD /kg CD /kg CD /kg CD /kg CD DR 10 6 /kg PBCD /l a Spearman s rank correlation. Table 3 Effect of CD34 + dose and PBCD34 + count on percent of patients with low blood counts after ASCT WBC Hemoglobin Platelets /l 120 g/l /l % patients % patients % patients CD34 + cells dose /kg /kg /kg /kg Overall P value PBCD34 + cell count /l /l /l /l Overall P value % Patients WBC Platelets Hb >350 PBCD34+ count ( 106/l) Figure 1 Percent of patients with low peripheral blood counts according to the peripheral blood CD34 + cell count on the first apheresis day. post-transplant period after ASCT. Amigo et al 12 previously showed that the CD34 + cell dose/kg correlates with both 6 and 12 month hemoglobin and platelets. No such effect on white cell count was observed. Hematopoiesis was not affected by alkylating agent use, age, prior radiotherapy, or diagnosis. Perez-Simon et al 13 also found an influence of CD34 + cells on long-term engraftment and observed that lower cell doses also correlates with transfusion and antibiotic requirements, febrile episodes and days of hospitalization post ASCT. In this study, we attempted to analyze factors that may influence long-term bone marrow function after ASCT. Less than one third of patients in our study had completely normal WBC, platelets and hemoglobin at a median of 6 months post ASCT. Our results confirm a strong association between CD34 + cells/kg infused and long-term engraftment. The number of circulating CD34 + cells in the peripheral blood has been shown to predict the number of CD34 + cells that can be collected by apheresis. 20,21 In this study, very good correlation was seen between the number of PBCD34 + cells as measured on the first apheresis day, and the total number of CD34 + cells/kg infused. The predictive strength of these two measures was very close, as may be expected from their close correlation with one another, and the differences did not reach statistical significance. The PBCD34 + cell count also correlated well with long-term engraftment by multivariate analysis, and seemed to be a better predictor of low WBC and Hb than was the infused CD34 + cell dose/kg. Patients with a PBCD34 + count above the median count of /l, or CD34 + cell doses above the median of /kg were most likely to have normal long-term peripheral blood counts (Table 3). The intermediate and lowest ranges on the other hand frequently produced cytopenias, although it is worth noting that low counts were not infrequent even in patients whose PBCD34 + count or CD34 + dose were above the upper quartile for the study population. Though both measures of CD34 + cell mobilization correlate with both early and late engraftment, rapid early hematopoietic recovery did not ensure normal long-term hematopoietic engraftment. The predictive value of CD34 + cells/kg and the peripheral blood CD34 + cell count remained statistically significant even after controlling for other variables in multivariate analysis. None of the other factors studied had a consistent effect by multivariate analysis on long-term engraftment. As shown in Table 2, CD34 + cell subsets, could, in some cases, correlate with long-term engraftment by univariate analysis. However, both the CD34 + cells/kg

5 and the PBCD34 + cell count correlated better than any of the CD34 + subsets. Only these two measures remained significant in multivariate analysis, with none of the subsets of cells continuing to be predictive. Several limitations to this study must be recognized. We retrospectively studied a heterogeneous patient population that received a number of different mobilization and chemotherapy regimens. Although we did not identify a significant relationship of any clinical factor with long-term hematopoiesis in multivariate analysis, we cannot conclude that one does not exist. We may simply have had too few study subjects to detect such an association. Another limitation is that in 15 patients, the follow-up blood counts were performed after confirmed relapse of their primary malignancy. Although no systemic chemotherapy had been administered from the time of ASCT until the 6 month CBC, the CBC for these patients may have been affected by disease recurrence. However, the relationship of the 6 month CBC to CD34 + dose or PBCD34 + count was independent of relapse (data not shown). Another problem was the variation in the timing of the follow-up CBC by the physicians following the patients after transfer back from the transplant unit. The majority of patients, however, did have a CBC measured between 5 and 7 months post ASCT. The cut-off values we used for peripheral blood counts represent the lower limit of normal range in our laboratory. These endpoints were chosen to reflect what would best be described as normal hematopoiesis, so that patients with any evidence of abnormal hematopoiesis could be identified and analyzed. It could be argued that the use of lower values to define these endpoints may have had more clinical relevance. However, even mild anemia that would not ordinarily require transfusion can adversely affect the quality of life of cancer patients. Quality of life improves up to a hemoglobin of 120 g/l, making a normal hemoglobin an important long-term goal following cancer therapy. 22,23 In addition, low blood counts may be associated with an increased replicative burden on existing stem cells, resulting in their rapid aging. One can theorize that this proliferative stress may possibly result in higher incidences of clonal disorders later in life. Such a theory is supported by reports of telomere shortening in peripheral mononuclear cells of patients who had undergone ASCT, and negative correlations with the number of mononuclear cells infused. 24 Further, Friedberg and colleagues 25 have recently reported a negative correlation between the infused cell dose and incidence of myelodysplasia after autologous bone marrow transplantation. The value of PBCD34 + cell count in predicting long-term hematopoietic outcome after ASCT has not previously been recognized. From our data, the PBCD34 + cell count seems to be at least as good as, or perhaps even a better predictor of long-term engraftment than the CD34 + dose/kg. This is particularly true considering that 21% of patients did not receive the entire collected product. The data were analyzed, however, based on the actual infused dose and not the collected dose. One might expect the infused dose to correlate better than the mobilized PBCD34 + count. The finding that the PBCD34 + count correlates better with longterm engraftment than the infused CD34 + dose may indicate that the PBCD34 + count is a marker of the quality of the stem cells harvested. It is possible that the ability to mobilize large numbers of CD34 + cells, as reflected by high PBCD34 + counts as apheresis starts, indicates the presence of a healthier marrow microenvironment or a healthier population of hematopoietic stem cells. Patients with low PBCD34 + cell counts, on the other hand, may have poorer functioning stem cells that are less capable of maintaining normal long-term hematopoiesis, or a marrow microenvironment that does not support normal marrow function. 26,27 Such a problem may not necessarily be overcome by collecting larger numbers of CD34 + cells in extra apheresis procedures. Furthermore, there may be some effect of cryopreservation and thawing on the final infused product that would diminish the capacity of poor quality products for long-term hematopoiesis more than superior products. Such variables would have no effect on the PBCD34 + cell count. We hypothesized that the quality of the autograft could be assessed by measuring subsets of CD34 + cells. Our study, however, demonstrated that none of the subsets analyzed were good surrogate markers of autograft quality. Long-term hematopoiesis may be dependent on a population of CD34-negative cells or some subset of CD34 + cells that we did not measure (ie true stem cells). 28,29 If this is true, the PBCD34 + cell count may better reflect this unmeasured cell population than the total CD34 + cell dose infused. In conclusion, our results suggest that the total CD34 + cell dose does predict for blood counts several months post ASCT, and that the CD34 + subsets we studied (41 +,33, 38,90 +,38 DR ) did not add useful information to the total CD34 + dose. Furthermore, we report for the first time that the peripheral blood CD34 + count ( 10 6 /l) on the first apheresis morning is perhaps an even better predictor of long-term hematopoiesis than the infused CD34 + dose/kg. This in turn suggests that the PBCD34 + count is a marker for collecting a high quality autograft, and that the quality of the autograft is not adequately assessed by the CD34 + subsets we measured. Finally, it is reported that most patients who receive the currently accepted minimum CD34 + dose for autografting ( CD34 + cells/kg) will have low blood counts at a median of 6 months post ASCT. Our study suggests that the risk of having prolonged low blood counts may not be minimized unless the CD34 + cell dose is CD34 + cells/kg. This result needs to be confirmed by other investigators. References 1 Stewart DA, Guo D, Luider J et al. Factors predicting engraftment of autologous blood stem cells: CD34 + subsets inferior to the total CD34+ cell dose. Bone Marrow Transplant 1999; 23: Meldgaard Knudsen L, Jensen L, Jarlbaek L et al. Subsets of CD34+ hematopoietic progenitors and platelet recovery after high dose chemotherapy and peripheral blood stem cell transplantation. Haematologica 1999; 84: Weaver CH, Hazelton B, Birch R et al. An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 1995; 86:

6 Pecora AL, Preti RA, Gleim GW et al. CD34+CD33 cells influence days to engraftment and transfusion requirements in autologous blood stem-cell recipients. J Clin Oncol 1998; 16: Olivieri A, Offidani M, Montanari M et al. Factors affecting hemopoietic recovery after high-dose therapy and autologous peripheral blood progenitor cell transplantation: a single center experience. Haematologica 1998; 83: Ketterer N, Salles G, Raba M et al. High CD34(+) cell counts decrease hematologic toxicity of autologous peripheral blood progenitor cell transplantation. Blood 1998; 91: Krause DS, Fackler MJ, Civin CI et al. CD34: structure, biology, and clinical utility. Blood 1996; 87: Buhring HJ, Asenbauer B, Katrilaka K et al. Sequential expression of CD34 and CD33 antigens on myeloid colonyforming cells. Eur J Haematol 1989; 42: Loken MR, Shah VO, Dattilio KL et al. Flow cytometric analysis of human bone marrow: I. Normal erythroid development. Blood 1987; 69: Debili N, Issaad C, Masse JM et al. Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation. Blood 1992; 80: Henon P, Sovalat H, Becker M et al. Primordial role of CD cells in early and late trilineage haemopoietic engraftment after autologous blood cell transplantation. Br J Haematol 1998; 103: Amigo ML, del Canizo MC, Caballero MD et al. Factors that influence long-term hematopoietic function following autologous stem cell transplantation. Bone Marrow Transplant 1999; 24: Perez-Simon JA, Martin A, Caballero D et al. Clinical significance of CD34 + cell dose in long-term engraftment following autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 1999; 24: Haas R, Witt B, Mohle R et al. Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support. Blood 1995; 85: Kiss JE, Rybka WB, Winkelstein A et al. Relationship of CD34 + cell dose to early and late hematopoiesis following autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 1997; 19: Stewart DA, Guo D, Morris D et al. Superior autologous blood stem cell mobilization from dose-intensive cyclophosphamide, etoposide, cisplatin plus G-CSF than from less intensive chemotherapy regimens. Bone Marrow Transplant 1999; 23: Terstappen LW, Huang S, Safford M et al. Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38 progenitor cells. Blood 1991; 77: Bernstein ID, Leary AG, Andrews RG et al. Blast colonyforming cells and precursors of colony-forming cells detectable in long-term marrow culture express the same phenotype (CD33- CD34+). Exp Hematol 1991; 19: Brandt J, Baird N, Lu L et al. Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro. J Clin Invest 1988; 82: Schots R, Van Riet I, Damiaens S et al. The absolute number of circulating CD34 + cells predicts the number of hematopoietic stem cells that can be collected by apheresis. Bone Marrow Transplant 1996; 17: Perez-Simon JA, Caballero MD, Corral M et al. Minimal number of circulating CD34+ cells to ensure successful leukapheresis and engraftment in autologous peripheral blood progenitor cell transplantation. Transfusion 1998; 38: Cleeland CS, Demetri GD, Glaspy J et al. Identifying hemoglobin level for optimal quality of life: results of an incremental analysis. J Clin Oncol 1999; 18: 2215 (Abstr.). 23 Glaspy J, Bukowski R, Steinberg D et al. Impact of therapy with epoetin alfa on clinical outcomes in patients with nonmyeloid malignancies during cancer chemotherapy in community oncology practice. Procrit Study Group. J Clin Oncol 1997; 15: Lee J, Kook H, Chung I et al. Telomere length changes in patients undergoing hematopoietic stem cell transplantation. Bone Marrow Transplant 1999; 24: Friedberg JW, Neuberg D, Stone RM et al. Outcome in patients with myelodysplastic syndrome after autologous bone marrow transplantation for non-hodgkin s lymphoma. J Clin Oncol 1999; 17: Bernstein ID, Andrews RG, Rowley S. Isolation of human hematopoietic stem cells. Blood Cells 1994; 20: Verfaillie CM. Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation. Blood 1993; 82: Lange C, Kaltz C, Thalmeier K et al. Hematopoietic reconstitution of syngeneic mice with a peripheral blood-derived, monoclonal CD34, Sca-1+, Thy-1(low), c-kit+ stem cell line. J Hematother Stem Cell Res 1999; 8: Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nature Med 1998; 4: