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Improved haematopoietic recovery following transplantation with ex vivo-expanded mobilized blood cells*

H. Miles Prince,1 Paul J. Simmons,2 Genevieve Whitty,1,2 Dominic P. Wall,1 Lesley Barber,1 Guy C. Toner,1 John F. Seymour,1 Gary Richardson,3 Robert Mrongovius4 and David N. Haylock1,2 1

Division of Haematology and Medical Oncology,

Peter MacCallum Cancer Center, 2Stem Cell Research Laboratory, Peter MacCallum Cancer Center, 3Department of Oncology, Cabrini Hospital, and 4Amgen Australia Pty Ltd, Melbourne, Australia Received 13 April 2004; accepted for publication 26 May 2004 Correspondence: Associate Professor H. Miles Prince, Division of Haematology and Medical Oncology, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett St East Melbourne, Victoria 8006, Australia. E-mail: [email protected] *Presented in abstract form (oral presentation) at the 45th Annual Meeting of the American Society of Hematology, San Diego, CA, 9

Summary Infusions of ex vivo-expanded (EXE) mobilized blood cells have been explored to enhance haematopoietic recovery following high dose chemotherapy (HDT). However, prior studies have not consistently demonstrated improvements in trilineage haematopoietic recovery. Three cohorts of three patients with breast cancer received three cycles of repetitive HDT supported by either unmanipulated (UM) and/or EXE cells. Efficacy was assessed by an internal comparison of each patient’s consecutive HDT cycles, and to 106 historical UM infusions. Twenty-one cycles were supported by EXE cells and six by UM cells alone. Infusions of EXE cells resulted in fewer days with an absolute neutrophil count (ANC) 0Æ1 · 109/l (median 5 vs. 8 d, P ¼ 0Æ0002). This resulted in a major reduction in the incidence of febrile neutropenia compared with UM cycles (0% vs. 83%; P ¼ 0Æ008) and in 66% of historical UM cycles (P ¼ 0Æ01) and a marked reduction in hospital re-admission. There were also fewer platelet transfusions required (43% vs. 100%; P ¼ 0Æ009). We conclude that EXE cells enhance both neutrophil and platelet recovery and reduce febrile neutropenia, platelet transfusion and hospital re-admission. Keywords: ex vivo, cell expansion, CD34, transplantation.

December 2003.

Randomized trials in the treatment of relapsed aggressive lymphoma and multiple myeloma have demonstrated that high dose chemotherapy (HDT) supported by autologous bone marrow or peripheral blood progenitor cells (PBPC) achieve higher response rates than conventional-dose chemotherapy and this translates into improved disease-free and overall survival (Philip et al, 1995; Linch et al, 1993; Attal et al, 1996; Child et al, 2003). However, the toxicity of such therapy is substantial, largely related to the period of obligate neutropenia and thrombocytopenia that follows the HDT regimen, with the associated high incidence of febrile neutropenia and the need for prophylactic platelet transfusions (British Committee for Standards in Haematology, Blood Transfusion Task Force, 2003). Various approaches have been explored to enhance haematopoietic recovery following HDT and reduce the severity and length of pancytopenia including the use of cytokine-mobilized peripheral blood which, when compared with bone doi:10.1111/j.1365-2141.2004.05081.x

marrow, results in more rapid neutrophil and platelet recovery (Schmitz et al, 1996). The administration of haematopoietic growth factors, such as granulocyte-colony stimulating factor (G-CSF), following PBPC infusion have also been demonstrated to further shorten the period of neutropenia by approximately 3 d (Lee et al, 1998; Linch et al, 1997). Nonetheless, all studies of PBPC transplantation report a period of severe ‘obligate’ thrombocytopenia with the time required for platelets to exceed 20 · 109/l without transfusion support generally 11–14 d after PBPC infusion. The period of obligate neutropenia with an absolute neutrophil count (ANC) to greater than 1Æ0 · 109/l is approximately 9–11 d (Bender et al, 1992; Bensinger et al, 1995; Weaver et al, 1995; To et al, 1997; Ketterer et al, 1998). It is well recognized that the number of immature PBPC infused, as measured by the CD34+ cell content (Sutherland et al, 1996) is critical to the rate of neutrophil and platelet recovery, with doses below 2 · 106/kg generally considered suboptimal and resulting in prolonged

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 536–545

Ex Vivo Expansion of Haematopoietic Cells cytopenias (Bender et al, 1992; To et al, 1997). However, there is conflicting evidence in the literature as to the optimum number of CD34+ cells required to minimize the period of these obligate cytopenias. Ketterer et al (1998) demonstrated a 2-d improvement in platelet recovery to >20 · 109/l when patients received more than 15 · 106/kg CD34+ cells compared with those receiving 5–15 · 106/kg CD34+ cells (8 d vs. 10 d) and a modest 1-d improvement in time to ANC >0Æ5 · 109/l (10 d vs. 11 d) (Ketterer et al, 1998). Weaver et al (1995) demonstrated that CD34+ cell doses in excess of 7Æ5 · 106/kg and 12Æ5 · 106/kg were optimal for rapid neutrophil and platelet recovery, respectively with the fastest median times to ANC >0Æ5 · 109/l and time to platelets >20 · 109/l both being 8 d. Conversely, Bensinger et al (1995) and To et al (1997) were unable to demonstrate any discernable difference in haematopoietic recovery in patients who received in excess of 5 · 106/kg CD34+ cells. Despite these discrepancies, irrespective of cell dose, patients almost universally require platelet transfusions because of severe thrombocytopenia. One proposed means of attempting to ameliorate these cytopenias is to transplant ex vivo-expanded (EXE) mobilized PBPC that have been propagated to promote the numerical expansion and differentiation of haematopoietic progenitors into neutrophil and megakaryocyte precursors (Devine et al, 2003). Once infused, these cells should then ‘home’ to the marrow and complete their maturation in vivo to produce mature functional end cells within a few days. Indeed, preclinical data indicates that both myeloid and megakaryocytic progenitors can be expanded in static culture systems with various growth factor combinations (Haylock et al, 1992; To et al, 1997). To date, several clinical ex vivo expansion studies based on this rationale have been performed, using a variety of culture systems and cytokine combinations (Reiffers et al, 1999; Williams et al, 1996; McNiece et al, 2000; Paquette et al, 2000; Zimmerman et al, 2000; Engelhardt et al, 2001; Reichle et al, 2003). Although a number of these studies have demonstrated a positive impact on myeloid cell recovery (with one demonstrating a modest impact on platelet recovery; Paquette et al, 2000) all except one study (Reichle et al, 2003) have utilized historical control patients. One approach to increase the anti-tumour effectiveness of HDT is to administer repeated cycles of HDT, each supported by PBPC. This strategy appears beneficial in selected patients with myeloma (Attal et al, 2003) and relapsed or high-risk germ cell tumours (Bokemeyer et al, 1999). Although the use of HDT remains controversial in the management of breast cancer, there is evidence that some patient groups may benefit and consequently, there is a general consensus that its use warrants ongoing investigation in the setting of novel clinical trials (Elfenbein, 2003; Tartarone et al, 2003). Indeed, in a recently reported randomized multicentre study on high risk breast cancer, repeated cycles of HDT were shown to be of benefit (Basser et al, 2003). We have been investigating the role of repetitive HDT in patients with advanced breast cancer,

delivered largely in the outpatient setting. However, the incidence of febrile neutropenia, thrombocytopenia and consequent hospital re-admission rates are high and we sought to explore the use of EXE PBPC to support such therapy (Prince et al, 1999, 2000a,b, 2002a). In designing this trial we took advantage of the fact that patients could be infused with both unmanipulated (UM) cells and EXE cells following separate cycles and thus could act as their own internal control. Thus differences in haematopoietic recovery between the two different forms of cell products could be directly compared.

Materials and methods Eligibility criteria Patients with histologically and/or cytologically proven advanced breast cancer were eligible for entry into this prospective trial. Patients were ineligible if they had received prior radiotherapy to >25% of marrow bearing areas, had an Eastern Cooperative Oncology Group (ECOG) performance status >2, an ANC 2 · 106/kg CD34+ cells). High-dose therapy was administered intravenously as 538

previously described and consisted of paclitaxel (175 mg/m2, given over 3 h on day )5), cyclophosphamide (4 g/m2, day )4) and thiotepa (300 mg/m2, days )4, )3 and )2) (Prince et al, 2002a,b) Cells were thawed and infused on day 0. All patients received filgrastim 5 lg/kg/d subcutaneously from day 1 posttransplant until the postnadir ANC was ‡1Æ5 · 109/l for three consecutive days. No prophylactic antibiotics were administered. An identical filgrastim and antibiotic policy was also applied in the historical control group. The planned interval between cycles of HDT was 35 d and patients proceed to the next cycle of HDT if they had not experienced unacceptable non-haematopoietic toxicity and the platelet count exceeded 70 · 109/l.

Patient monitoring post-HDT and infusion Patients were admitted to hospital on the day of haematopoietic cell infusion and were discharged the following day. Daily complete blood analysis was scheduled for the first 14 d following transplantation. Prophylactic platelet transfusions (5 units of pooled random donor platelets, or apheresis singledonor platelets in patients refractory to pooled platelets) were administered when the platelet count fell below 10 · 109/l or below 20 · 109/l if the patient had fever or was receiving intravenous amphotericin or other medications that altered platelet function. When febrile neutropenia occurred, patients were routinely admitted for intravenous broad-spectrum antibiotics. All these practices were consistently applied during the trial and the period during which the historical controls were treated.

Historical controls From our database, we selected consecutive patients who had received the same repetitive HDT for the treatment of advanced breast cancer and had received a minimum of 2 · 106/kg CD34+ cells per HDT cycle. We excluded patients whose PBPC had undergone CD34+ selection (Prince et al, 2002a,b). Unlike the current study, daily blood counts were not routinely performed in this group, but rather daily counts if an inpatient, otherwise a minimum of second daily counts until platelet recovery to >20 · 109/l.

Statistical analysis The duration of cytopenia was measured from the time of cell infusion (day 0) until the time when ANC exceeded 0Æ1 · 109/l and 1Æ0 · 109/l and platelets exceeded 20 and 50 · 109/l. Similarly, time to last platelet transfusion and spontaneous rise in platelets (first day when platelets rose without platelet transfusion) were measured from day 0. The proportions of patients recovering to those thresholds were estimated using the Kaplan–Meier method. For historical control patients, 98% had consecutive daily blood counts to an ANC >1Æ0 · 109/l and 92% had consecutive daily blood counts to platelet


Ex Vivo Expansion of Haematopoietic Cells recovery >20 · 109/l. For count recovery endpoints beyond this time, we utilized second-daily counts. Comparison of haematopoietic recovery parameters of patients transplanted with either UM PBPC, or EXE cells and historical controls were compared using the log-rank test. In all cases, the analysis was stratified for the dependent variable of the same patient receiving multiple cycles of HDT. This stratification was used for haematopoietic recovery measurements of study cycles supported by EXE cells and UM cells, and comparisons with the 43 patients in the control population. All categorical variables were analysed using chi-square statistics. A paired t-test was also used to compare haematopoietic recovery between EXE and UM cycles of HDT for inter-individual cycles and a nonparametric Mann–Whitney U-test to compare days of neutropenia and thrombocytopenia between the EXE, UM and historical (UM) groups. All results are expressed as two-sided P values; P < 0Æ05 were considered significant.

Results Patient characteristics The patient characteristics of the nine patients are detailed in Table I. Forty-three sequential historical control patients, who received a total of 106 of the same HDT cycles supported by UM PBPC, were identified and had received a median of 3Æ5 · 106/kg (range: 2Æ2–9Æ6 · 106/kg) CD34+ cells infused. Summary statistics did not suggest any substantial difference between the two groups on the various disease parameters.

Mobilization, apheresis and selection of CD34+ cells Given our previous experience in supporting repetitive HDT for breast cancer with UM PBPC or Isolex selected CD34+ cells (Prince et al, 2002a), we aimed to collect at least 2 · 106 CD34+ cells/kg for each UM PBPC graft. Furthermore, in accord with our prediction for the number of EXE cells required to improve haematopoietic recovery and our experience of preclinical ex vivo expansion cultures with mobilized blood CD34+ cells stimulated with G-CSF, SCF and PEGrHuMGDF (Haylock et al, 1992), we aimed to collect sufficient cells such that once processed on the Isolex 300i CD34selection device (see below) and then divided into equal aliquots, we could initiate each ex vivo expansion culture with at least 100 · 106 CD34+ cells. Five patients required only one mobilization cycle while the other four patients required a second mobilization with docetaxel/filgrastim. For the entire nine patients, a median of 7Æ2 · 106 CD34+ cells/kg was collected (range: 2Æ38–11Æ95 · 106/kg). For Isolex 300i processing, the apheresis products contained 4Æ16 ± 1Æ3 · 1010 (mean ± SD; range, 2Æ4–6Æ7 · 1010) leucocytes with an initial CD34+ cell frequency of 0Æ77 ± 0Æ44% (mean ± SD; range, 0Æ32–1Æ3%). CD34-selection with Isolex 300i processing yielded 295Æ5 ± 171Æ1 · 106 CD34+ cells (mean ± SD, range 89Æ3–621 · 106) at 94Æ4 ± 5Æ28% CD34+ cell purity (mean ± SD; range 81–98%) and 87Æ2 ± 16Æ5% (mean ± SD, range 45Æ1–100%) yield of CD34+ cells. After CD34-selection, we were able to obtain a median of 183Æ5 · 106 CD34+ cells (range: 85–273 · 106) to inoculate the ex vivo cultures.

Table I. Patient characteristics.

Characteristic Median age [years (range)] Unresectable locally advanced disease Metastatic disease Median number of sites (range) 1Æ0 · 109/l was 2 d faster (median 8 d vs. 10 d, P ¼ 0Æ005). In the historical control group, the median number of days with ANC 0Æ1 · 109/l of 9 d (range: 6–21 d, P < 0Æ0001) and >1Æ0 · 109/l of 10 d (range: 8–26 d, P < 0Æ0001; Table II) There were no episodes of late graft failure. None of the 21 cycles supported by EXE cells were complicated by febrile neutropenia, whereas five of the six (83%) transplant episodes using only UM PBPC were complicated by febrile neutropenia (P ¼ 0Æ008). When febrile neutropenia occurred following cycles supported by UM

PBPC, it lasted for a median of 5 d (range: 3–9 d). Similarly, the incidence of febrile neutropenia in the 106 cycles of HDT in the control group was 66% with a median duration of 4 d (range: 1–9 d), which was significantly different from that observed in the trial population receiving HDT supported by EXE cells (P ¼ 0Æ01). The impact on febrile neutropenia after infusion of EXE cells translated into a marked reduction in the need for hospital admission with none of the patients receiving cycles supported by EXE cells requiring re-admission, as compared with an 83% re-admission rate following cycles supported by UM cells alone (P ¼ 0Æ008). In the historical group, where all patients received UM cells, there was a similar rate of re-admission (78%) and for those admitted, there was a median hospital stay of 6 d (range:1–20 d), which was significantly different from that observed in the trial population receiving HDT supported by EXE cells (P ¼ 0Æ003; Table II). The HDT cycles supported by EXE cells required fewer platelet transfusions with nine cycles (43%) not requiring platelet support with median number of platelet transfusions of one per cycle (range: 0–3). In comparison, all of the six HDT cycles supported by UM PBPC alone required platelet transfusion, with a median of 3 (mean ¼ 2Æ6) platelet transfusions per cycle (range: 1–5) (P ¼ 0Æ009). Platelet nadirs were also assessed. In the UM group, with the exception of one patient who had a platelet transfusion at 19 · 109/l (sepsis and bleeding), the median platelet nadir was 7 · 109/l (range: 2–10, SD ¼ 3Æ0) as compared with the EXE group with a median nadir of 8 · 109/l (range: 4–17, SD ¼ 4Æ0) (P ¼ 0Æ03). The significant difference was attributable to the nine cycles with a platelet nadir above the transfusion threshold of 10 · 109/l. Patients in the historical control group required a median of three platelet transfusions (range: 1–29) per cycle. Furthermore, the median time to last platelet transfusion was shorter for the cycles supported by EXE cells than those supported by

Table II. Haematopoietic recovery following HDT cycles.

Days with ANC 0Æ1 · 109/l Days to ANC >1Æ0 · 109/l Incidence of febrile neutropenia (%) Median days of FN (range) Hospital re-admission for FN (%) Median days for hospital re-admission (range) Requirement for any platelet transfusions (%) Median number of platelet transfusions (range) Days to last platelet transfusion Days to spontaneous rise in platelets Days to platelets >20 · 109/l Days to platelets >50 · 109/l

Ex vivo-expanded (n ¼ 21) (range)

Unmanipulated (n ¼ 6) (range)

2 5 8 0 0 0 0 57 1 7 8 12 16

4 8 10 83 5 83 5 100 3 9 11 12 16

(0–5) (3–8) (6–12) (0)

(0–3) (0–11) (7–16) (7–17) (11–22)

(2–7) (8–9) (8–12)

P-value 0Æ002 0Æ0002 0Æ005 0Æ008

(3–9) 0Æ008 (0–9) 0Æ009 (1–5) (8–13) (8–16) (8–14) (11–18)

0Æ02 0Æ04 0Æ6 0Æ6

Historical cycles (n ¼ 106) (range) 6 9 10 66 4 78 6 100 3 11 14 14 19

(2–19) (6–21) (8–26)

P-value* 0Æ013 50 · 109/l was 19 d (range: 11–68 d) (P ¼ 0Æ03; Table II). There were no episodes of late thrombocytopenia (secondary graft failure). With respect to differences in haematopoietic recovery as patients progressed through the three cycles of HDT, we did not observe any difference in time to ANC or platelet recovery with successive cycles supported by EXE cells (cohort 3), or did we find any difference in recovery between cohorts 1 and 2 when receiving UM cells in cycle 1 and cycle 3, respectively (data not shown).

Discussion

Fig 2. Kaplan–Meier estimates of hematopoietic recovery. (A) Time to ANC >0Æ1 · 109/l. Median time to recovery for cycles supported by ex vivo-expanded cells (EXE) (n ¼ 21; dash-dot line) was 5 d, unmanipulated (UM) cells alone (n ¼ 6; solid line) was 8 d, and historical control group with UM cells (n ¼ 106; dotted line) was 9 d. P ¼ 0Æ0002 between EXE and UM and P < 0Æ0001 between EXE and historical group. (B) Time to day of last platelet transfusion. Kaplan– Meier estimates of median time to recovery for cycles supported by EXE cells (n ¼ 21; dash-dot line) was 7 d versus UM cells alone (n ¼ 6; solid line) of 9 d (P ¼ 0Æ02), and historical group (n ¼ 106; dotted line) of 11 d (P < 0Æ0001 compared with EXE cells).

This study demonstrated that CD34+ cells expanded ex vivo with the cytokine combination of G-CSF, SCF and PEGrHuMGDF resulted in a 33-fold and 2Æ8-fold expansion of total cells and CD34+ cells respectively. The most important effect demonstrated following infusion of these cells was the markedly reduced depth of thrombocytopenia following HDT, which resulted in 43% of HDT cycles not requiring any platelet transfusions. Another major finding was that transplantation of an EXE product reduced the depth and length of neutropenia, with a consequent reduction in episodes of febrile neutropenia. Although the HDT regimen used in this


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H. M. Prince et al study is not widely utilized, we have previously demonstrated that this, and similar regimens, are extremely myelosuppressive as demonstrated by prolonged cytopenias if inadequate doses of CD34+ cells are infused (Prince et al, 1999, 2000a,b, 2002a). Indeed, the kinetics of haematopoietic recovery with this HDT regimen are very similar to those of other regimens used for the treatment of diseases such as lymphoma, germ cell tumours and myeloma and thus a good model to examine the effectiveness of ex vivo expansion strategies. Improvement in neutrophil recovery was such that HDT cycles supported by EXE cells achieved shallower neutrophil nadirs and shorter periods of neutropenia compared with those cycles supported by UM cells. The utilization of ‘internal’ controls, with six patients having one of their three HDT cycles supported by UM cells with the remaining two cycles supported by EXE cells, provides compelling evidence that the EXE cells enhanced neutrophil recovery. Indeed, in most cases, the CD34+ cell content in the infused UM product was similar or higher than the starting CD34+ cell content of the EXE products. The finding of enhanced neutrophil recovery in the EXE cycles is further supported by the data provided by the historical control group; this comparison also demonstrated a substantial enhancement of neutrophil recovery with the infusion of EXE cells. Of note, the CD34+ cell doses infused in the historical controls all exceeded 2 · 106/kg; doses that are recognized to result in rapid and reliable haematopoietic recovery (To et al, 1997). The consequence of this enhanced neutrophil recovery was a significant reduction in the incidence of febrile neutropenia. Indeed, none of the patients who received HDT supported by EXE cells experienced this complication during those cycles. In contrast, the incidence of febrile neutropenia after HDT cycles supported by UM cells alone was 83%. In turn, the re-admission rate for febrile neutropenia was 0% following infusion of EXE cells, as compared with 83% in this trial, and 78% in the historical control group. Improvements in platelet recovery are harder to demonstrate, largely because of the requirement for prophylactic platelet transfusions, which is further complicated by the need to alter the prophylaxis threshold according to the patients clinical state, and the inherent variability of platelet increments following infusion. Thus, end points, such as time to platelets >20 · 109/l, are difficult to quantify and the number of platelet transfusions used may reflect more than just the haematopoietic recovery of the patient. Notwithstanding these caveats, we have demonstrated that in 43% of cycles supported by EXE cells, platelet transfusions were not required, a result of a shallower platelet nadir. This finding contrasts to the platelet requirements in the cycles supported with UM cells, both in study patients and historical controls, where all patients required platelet transfusions. The findings of reduced platelet utilization and time to a spontaneous rise in platelet count support the above observation and taken together, strongly support the contention that the use of EXE cells impact platelet recovery. This encouraging data hopefully will lead the way to 542

further exploration of modifications of this expansion strategy so that nadirs above the platelet transfusion threshold are more consistently achieved. It could be argued that the improved haematopoietic recovery was simply the result of the additional number of CD34+ cells in the starting population of the ex vivo cultures, which, when combined with UM PBPC, resulted in a superior dose of progenitors at the time of infusion. We have no unequivocal evidence to disprove this notion but consider it unlikely. First, the median number of CD34+ cells to initiate cultures was relatively modest at 2Æ76 · 106/kg and although there is some data that would indicate that increasing the infused CD34+ cell dose speeds haematopoietic recovery, such doses are in the range of 12Æ5 · 106/kg or above (Weaver et al, 1995; Ketterer et al, 1998). Secondly, statistical analysis has demonstrated no correlation between dose of EXE CD34+ cells infused and the rate or time of either neutrophil or platelet recovery (data not shown). To help address this issue, we would have liked to have performed a comparison of haematopoietic recovery in cycles supported by UM versus EXE cells infused with equivalent CD34+ cell numbers. However, a direct comparison was not possible because in the cycles supported by UM PBPC, the maximum dose of infused CD34+ cells was 4Æ1 · 106/kg, but following expansion, only two of 21 cultures had CD34+ cell counts that fell within this range (median infused dose of 6Æ29 with range of 1Æ51–20Æ6 · 106/kg). Although further studies may help to determine the relative importance of CD34+ cell dose in expanded cell products, we consider it more likely that the 33-fold total cell expansion in culture led to the enhanced haematopoietic recovery. Nonetheless, our results are important in that they demonstrate that the most immature haematopoietic progenitors, as enumerated by the CD34+ content, are not only sustained but have a median of 2Æ8-fold expansion and this CD34+ expansion may be particularly important as we explore ex vivo expansion for cord blood, or for patients with suboptimal PBPC yields. In our previous studies, we demonstrated that functional impairment of the microenvironment occurred with repeated cycles of HDT, and the consequence of this was delayed haematopoietic recovery (Prince et al, 1999, 2000a,b, 2002a). However, we had demonstrated that this effect only had a marginal influence on platelet recovery, and only affected neutrophil recovery if suboptimal doses of CD34+ cells were infused. Indeed, in this current study we observed improvements in both neutrophil and platelet recovery in cohort 1, where UM cells were infused in cycle 1 and EXE in cells in the cycles two and three, and thus the observation of improved haematopoietic recovery in the face of accumulating microenvironmental damage indicates the potency of the expanded cells. Moreover, we did not observe progressive slowing of either neutrophil or platelet recovery as patients progressed through their three cycles of HDT supported with EXE cells (cohort 3). This would be in concordance with our previous findings that the impact of progressive microenvironmental


Ex Vivo Expansion of Haematopoietic Cells damage only becomes clinically apparent when patients are infused with suboptimal doses of CD34+ cells (Prince et al, 2002b). Other studies have attempted to examine the impact of ex vivo expansion on haematopoietic recovery. These studies have utilized various combinations of cytokines, used different methods of cell expansion and investigated patients with various disease types (Reiffers et al, 1999; Williams et al, 1996; McNiece et al, 2000; Paquette et al, 2000; Zimmerman et al, 2000; Engelhardt et al, 2001; Devine et al, 2003; Reichle et al, 2003). The studies by Reiffers et al (1999), Paquette et al (2000) and McNiece et al (2000), all utilized the same cytokine combination as in our study and they too demonstrated an impact on neutrophil recovery; however interpretation of the data is hampered because their comparators were historical controls. Only Paquette et al (2000) demonstrated an impact on platelet recovery when compared with historical controls, and the inability of the other investigators to consistently demonstrate an impact on platelets may partly be the result of the use of historical controls or possibly differences in initiating cell concentrations, media and the washing process, which all may have influenced megakaryocyte precursor frequency, viability and function. To date there is only one study (using a different combination of cytokines) that utilizes internal controls to assess the impact of EXE cells (Reichle et al, 2003). They demonstrated an impact of EXE cells on myeloid recovery and infectious complications but did not report on platelet recovery (Reichle et al, 2003). Our study does have limitations. Although we used internal comparisons of haematopoietic recovery as the primary means of assessing the impact of ex vivo expansion, we too used historical controls to support these data. Given, the inherent deficiencies of using a historical control group (not least the lack of daily blood counts in the historical group, thus making estimations of haematopoietic recovery somewhat imprecise), we feel the comparative findings on haematopoietic recovery are supportive at best. Nonetheless, the data the historical group provides on re-admission rates is compelling. Furthermore, the cohort of nine patients were able to obtain sufficient PBPC to initiate the ex vivo culture. Thus our data may not necessarily be applied to patients with low PBPC yields, an area that we believe deserves further investigation. This study examined the effect of ex vivo expansion in patients with breast cancer, a disease where the role of HDT remains controversial. Although, recent studies have demonstrated the potential benefit of HDT for these patients (Basser et al, 2003; Rodenhuis et al, 2003), the question remains – can our data be applied to other diseases in which HDT is standard of care? The answer is probably ‘no’ as the regimens used for those diseases have additional non-haematopoietic toxicities, such as severe mucositis and gastrointestinal toxicities, where improvement in neutrophil and platelet recovery may not impact as dramatically on hospital length of stay or re-admission rates.

Our results demonstrate for the first time that EXE cells can substantially improve both neutrophil and platelet recovery and this translated into meaningful clinical benefits. This proof-of-principle demonstration therefore allows us to enthusiastically pursue further optimization of culture processing (such as various cytokine combinations and bioreactor processing devices that are less labour intensive), and then to explore ex vivo expansion strategies in other diseases that routinely utilize HDT and autologous PBPC transplantation.

Acknowledgements We would like to acknowledge the contributions of Ms Luiza Mints-Kotowska and Dr Lawrence Teoh for assistance in the stem cell processing laboratory and collection of historical data, Ms Dianne Tomita (Amgen Inc.), and Mr Jamie MacMillan (Amgen Australia Pty Ltd) for review of statistical analysis, and the referring physicians for their ongoing care, and ward and ambulatory care nurses and allied health staff for their excellent patient management. This was an investigator-driven study and was supported in part by a grant-in-aid by Amgen Australia Pty Ltd.

Financial disclosure One of the authors (R.M.) is employed by a company (Amgen, Australia Pty Ltd) whose product was studied in the present work.

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