M. R. Nowrousian*, S. Waschke, P. Bojko, A. Welt, P. Schuett, P. Ebeling, M. Flasshove, T. Moritz, J. Schuette & S. Seeber

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1 Symposium article Annals of Oncology 14 (Supplement 1): i29 i36, 2003 DOI: /annonc/mdg706 Impact of chemotherapy regimen and hematopoietic growth factor on mobilization and collection of peripheral blood stem cells in cancer patients M. R. Nowrousian*, S. Waschke, P. Bojko, A. Welt, P. Schuett, P. Ebeling, M. Flasshove, T. Moritz, J. Schuette & S. Seeber Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Essen Medical School, Essen, Germany Introduction Various chemotherapy regimens, combined with recombinant human granulocyte colony-stimulating factor (rhg-csf) or recombinant granulocyte macrophage CSF (rhgm-csf) are used in cancer patients to mobilize and collect peripheral blood stem cells (PBSC). In this retrospective study, we evaluated and compared the efficacy of such regimens in 262 patients with different types of malignant diseases. The following chemotherapy regimens were applied: ifosfamide etoposide cisplatin or bleomycin (n = 96; mainly patients with testicular cancer); ifosfamide etoposide plus or minus cytosine arabinoside (Ara-C) or vincristine (VCR) (n = 52; mainly patients with lymphoma); cyclophosphamide anthracycline (n = 53; mainly patients with breast cancer); intermediate to high dose (ID HD) cyclophosphamide (n = 37; mainly patients with breast or ovarian cancer, or multiple myeloma; and others (n = 24). rhg-csf or rhgm-csf, each at an average daily dose of 5 µg/kg body weight, were used in 166 and 96 patients, respectively. The study evaluated and compared the efficacy of these two cytokines. In patients receiving rhg-csf, CD34+ cells could be collected earlier (median: day 14 versus day 16) and there was a significantly higher white blood cell count (WBC) (median versus 5550/µl) and CD34+ cell count (median 88 versus 43/µl) at the start of apheresis, and a significantly higher CD34+ cell yield (median versus /kg) than in patients who received rhgm-csf. Among the various chemotherapeutic regimens used, each combined with rhg-csf, ifosfamide etoposide plus or minus Ara-C or VCR mobilized a significantly higher number of CD34+ cells (median 119/µl) and produced a significantly higher harvest of these cells (median /kg) than cyclophosphamide anthracycline (median 87/µl and /kg, respectively) or ID HD cyclophosphamide (median 59/µl and /kg, respectively). Ifosfamide etoposide plus or minus Ara-C or VCR was also superior to ifosfamide etoposide cisplatin or bleomycin (median 78/µl and /kg, respectively), but at borderline significance. The outcome of PBSC mobilization and collection appeared to be negatively influenced by the number of relapses before the current salvage treatment. These data indicate that mobilization and collection of PBSC strongly depend on the type of hematopoietic growth factor and chemotherapeutic regimen used. The data further show rhg-csf is a more effective growth factor than rhgm-csf and ifosfamide etoposide-based regimens, particularly ifosfamide etoposide plus or minus Ara-C or VCR, are highly effective regimens in mobilizing and collecting CD34+ cells. In cancer patients, peripheral blood stem cells (PBSC) are usually mobilized by the application of chemotherapy and recombinant human granulocyte or granulocyte macrophage colonystimulating factor (rhg-csf and rhgm-csf, respectively). Among the chemotherapy regimens used, cyclophosphamide and etoposide in intermediate to high doses (ID HD), either as single agent or in combination with other drugs, have been reported to be particularly effective [1 22], as well as HD etoposide, even *Correspondence to: Dr M. R. Nowrousian, Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Essen Medical School, Hufelandstrasse 55, Essen, Germany. Tel: ; Fax: ; nowrousian@uni-essen.de Dedicated to Prof. Dr C. G. Schmidt on the occasion of his 80th birthday. in patients who have failed to mobilize PBSC with ID HD cyclophosphamide [18, 19, 21]. ID HD cyclophosphamide or etoposide, however, may be associated with substantial toxicities [10, 12, 19, 23], and in some malignant diseases, cyclophosphamide or etoposide as single agents may not be the appropriate treatment, even in ID HD [24]. Conventional doses (CD) of these drugs have been used in combination with other agents [8, 25 42]. Another effective agent in mobilizing PBSC is ifosfamide, which is frequently used together with other drugs, particularly etoposide [25 44]. Many regimens have been reported to be useful in mobilizing PBSC [18, 21, 22, 33 40, 42 44], but there are a limited number of studies comparing the efficacy of different drugs or drug combinations [4, 7, 8, 14, 16, 20, 25, 32]. In addition, there is no study comparing the efficacy of the frequently used ID HD cyclophosphamide, ifosfamide etoposide-based and CD cyclophosphamide regimens together 2003 European Society for Medical Oncology

2 i30 with an anthracycline. The aim of the present study was to make such a comparison using the same apheresis technique and the same target yield of PBSC. Peripheral blood stem cells can be mobilized using rhgm- CSF, rhg-csf, chemotherapy alone or chemotherapy followed by one of the two hematopoietic growth factors [45]. While rhg- CSF as single agent has been reported to be superior to rhgm- CSF [46, 47], the results of studies using chemotherapy and rhg-csf or rhgm-csf are controversial [49 51]. In two studies, rhg-csf was found to mobilize significantly more PBSC than rhgm-csf [50, 51], but in another study, the two factors appeared to have similar activity [49]. The present study therefore evaluated and compared the efficacy of these two cytokines. Further aims of the study were to investigate additional factors that could have an impact on mobilization and collection of PBSC and to define pre-apheresis factors that could help to calculate the yield of these cells and the amount of the blood volume that should be processed to collect a particular target yield. Patients and treatment Table 1. Patients Malignancy No. of patients Median age, years (range) Sex (M/F) Germ cell cancer (15 53) 98/1 Hodgkin s disease (16 54) 11/5 Non-Hodgkin s lymphoma (17 62) 24/22 Breast cancer (23 53) 0/70 Ovarian cancer (20 63) 0/13 Multiple myeloma (31 63) 7/11 Total (15 63) 140/122 Table 2. Chemotherapy regimens and drug doses used to mobilize CD34+ cells From March 1993 to June 1997, 262 patients received chemotherapy and rhg-csf or rhgm-csf to mobilize and collect PBSC (Tables 1 and 2). The following chemotherapy regimens were used: ifosfamide and etoposide plus cisplatin or bleomycin (n = 96; 36.6%), mainly in patients with germ cell cancer; ifosfamide and etoposide with or without cytosine arabinoside or vincristine (n = 52; 19.8%), mainly in patients with lymphoma; cyclophosphamide and doxorubicin or epirubicin (cyclophosphamide anthracycline) (n = 53; 20.2%), mainly in patients with breast cancer; ID HD cyclophosphamide (n = 37; 14.1%), mainly in patients with breast cancer, ovarian cancer or multiple myeloma; and other drug combinations (n = 24; 9.2%) (Tables 1 and 2). rhg-csf (filgrastim; Amgen, Munich, Germany) or rhgm-csf (molgramostim; Essex, Munich, Germany), each at an average daily dose of 5 µg/kg body wt, were given subcutaneously as hematopoietic growth factor in 166 and 96 patients, respectively. They were usually started 2 3 days after the last chemotherapy dose and continued until completion of PBSC collection. Stem cell monitoring and collection Hematopoietic stem cells were determined as CD34+ cells using flow cytometry analysis carried out on a Coulter Epics XL (Coulter, Hialeah, FL, USA). The cells were analyzed in a gate defined by CD45 antigen expression. Mouse isotype control (IgG1*PE; Becton Dickinson, San Jose, CA, USA) was used to account for unspecific fluorescence. To quantify the CD34+ cells, the following fluorochrome-conjugated monoclonal antibodies were used: CD34(Anti-HPCA-2)*PE (Becton Dickinson) and CD45(J33)*FITC (Immunotech, Marseille, France). About cells were stained with the appropriate amount of fluorochrome-conjugated antibody and incubated for 20 min at 4 C in the dark. Red blood cell lysis and leukocyte fixation were done with a Coulter Q-Prep Workstation and the ImmunoPrep reagent system (Coulter). Fifty thousand events were acquired for analysis. The total yield of each subset was calculated by multiplying the cell concentration with the collected blood volume after each blood volume separated [11]. Daily CD34+ cell monitoring from patients peripheral blood samples was started on day 10 after initiation of mobilization chemotherapy and PBSC collection was carried out when the peak concentration of CD34+ cells was expected to be reached, allowing the collection of a target number of CD34+ cells/kg body wt using a single apheresis. The target yield chosen had the aim of supporting two cycles of high-dose chemotherapy, each with at least CD34+ cells/kg body wt, and to provide the same amount of CD34+ cells as back-up. A Spectra blood cell separator (Gambro BCT, Lakewood, CO, USA) was used to collect the stem cells. Blood was drawn and returned either via peripheral veins or a double lumen Shaldon catheter. The patients blood volume was calculated based on weight, height and gender. Anticoagulation was done with citrate dextrose formula A (ACD-A; Baxter, Deerfield, IL, USA) and heparin (Hoffmann-La Roche, Grenzach-Wyhlen, Germany). The ACD-A to whole-blood ratio was set between 1:28 and 1:18, depending on the patients platelet counts, and heparin was added at 6 U/ml ACD-A. Calcium was replaced orally if symptoms of hypocalcemia occurred and platelet transfusions were given if indicated to keep the platelet count /µl at the start of apheresis. The whole-blood flow rate was set between 100 and 150 ml/min [11]. Regimen Drug doses per cycle Ifosfamide etoposide cisplatin or bleomycin Ifosfamide 6000 mg/m 2, etoposide 500 mg/m 2, cisplatin 100 mg/m 2 or bleomycin 30 mg total Ifosfamide etoposide ± cytosine arabinoside (Ara-C) or vincristine (VCR) Ifosfamide 7500 mg/m 2, etoposide mg/m 2, Ara-C 500 mg/m 2 or VCR 1 mg/m 2 Cyclophosphamide anthracycline Cyclophosphamide 1200 mg/m 2, doxorubicin 60 mg/m 2 or epirubicin 90 mg/m 2 Intermediate to high dose cyclophosphamide Cyclophosphamide: 2000 mg/m 2 in breast cancer; 2500 mg/m 2 in ovarian cancer; 6000 mg/m 2 in multiple myeloma

3 Results For the entire group of patients, the median day of starting CD34+ cell collection was day 15 after the beginning of chemotherapy. The median leukocyte and CD34+ cell counts in the peripheral blood were /µl and 70/µl, respectively, on the first day of apheresis and the median number of CD34+ cells collected was CD34+ cells/kg body wt. The median volume of blood processed was 17.3 l, equal to a median of 4.1 times the patients blood volume (Table 3). In 164 (62.6%) patients, only one apheresis was performed yielding a median of CD34+ cells/kg body wt. In 71 (27.1%) and 27 (10.3%) patients, one or two additional aphereses were required to achieve an appropriate yield. The number of CD34+ cells harvested declined on day 2 and day 3 of apheresis, reaching a median of /kg body wt and /kg body wt, respectively. For the complete apheresis, the median number of CD34+ cells collected was /kg body wt. The target yield of CD34+ cells/kg body wt was achieved in 55.3% of patients reaching a median harvest of CD34+ cells/kg body wt. The median harvest was CD34+ cells/kg body wt in 74.5% of patients and CD34+ cells/kg body wt in 89.7%. Predictive value of WBC and peripheral blood CD34+ cell count To investigate the potential role of WBC count and peripheral blood CD34+ cell count as predictive parameters for the yield of CD34+ cells, both variables were correlated with the amount of CD34+ cells collected from each microliter of blood on the first day of apheresis. The latter was calculated by dividing the total number of CD34+ cells collected by the total volume of the blood processed. The aim of this approach was to establish a statistical relationship that would allow one to predict the amount of CD34+ cells that could be collected individually in each patient at a certain WBC or CD34+ cell count in the peripheral blood and the volume of the blood that should be processed in order to collect a certain target amount of CD34+ cells/kg body wt. From the two parameters evaluated, the concentration of CD34+ cells in the peripheral blood showed a significant correlation Table 3. Results of PBSC mobilization and collection on the first day of apheresis in the entire group of 262 patients Median Range Day of 1st PBSC collection WBC at 1st PBSC collection (10 3 /µl) at 1st PBSC collection Processed blood volume (l) blood processed CD34+ cell collection efficacy a CD34+ cell yield (10 6 /kg body wt) a The ratio of blood processed to blood. PBSC, peripheral blood stem cell; WBC, white blood cell count. i31 (r = 0.817, P = ) with the yield of these cells (Figure 1). The median collection efficiency, defined as the amount of CD34+ cells collected from each microliter of blood, was 0.36 (Table 3) on the first day of apheresis, but declined to a median of 0.31 and 0.24 on the second and third day, respectively. No correlation was found between the leukocyte count in the peripheral blood and the number of CD34+ cells collected (r = 0.08, P = 0.20). Comparison between rhg-csf and rhgm-csf In patients receiving rhg-csf, it was possible to collect CD34+ cells earlier and there was a significantly higher leukocyte and CD34+ cell count in the peripheral blood at the start of apheresis and a significantly higher CD34+ cell yield than in patients who received rhgm-csf (Table 4). Similar results were obtained when the comparison was made in a homogenous group of patients regarding sex, age, malignant disease and chemotherapy regimen, such as patients with testicular cancer who exclusively received the drug combination ifosfamide etoposide cisplatin with rhg-csf or rhgm-csf (Table 4). Comparison between chemotherapy regimens In order to have comparable groups of patients regarding the use of hematopoietic growth factor, the impact of chemotherapy on CD34+ cell mobilization and collection was evaluated in patients who received rhg-csf. Among the chemotherapy regimens applied, ifosfamide etoposide plus or minus cytosine arabinoside (Ara-C) or vincristine (VCR) mobilized a significantly higher number of CD34+ cells and produced a significantly higher harvest of these cells than cyclophosphamide anthracycline (Table 5) or ID HD cyclophosphamide (Table 6). The cyclophos- Figure 1. Relationship between the absolute number of in the peripheral blood on the first day of apheresis and the corresponding amount of CD34+ cells collected. The median collection efficiency was 0.36, indicating that, for example, in a patient weighing 70 kg, 14.6 l blood had to be processed to achieve a CD34+ cell harvest of cells/kg body wt at a CD34+ cell concentration of 100/µl in the peripheral blood [(7.5 70):( ) = 14.6 l].

4 i32 Table 4. Comparison between rhg-csf and rhgm-csf in mobilizing and collecting PBSC rhg-csf rhgm-csf All patients (n = 166) PEI (n = 20) All patients (n = 96) PEI (n = 71) Day of 1st PBSC collection 14 a (9 26) 16 (13 20) 16 (5 23) 16 (5 23) WBC at 1st PBSC collection (10 3 /µl) 11.4 a ( ) 10.0 a ( ) 5.6 ( ) 5.3 ( ) at 1st PBSC collection 88 a ( ) 78 a ( ) 43 (7 774) 45 (10 774) blood processed 30 a ( ) 30 (4 210) 16 ( ) 17 (3 201) CD34+ cell yield (10 6 /kg body wt) 7 a ( ) 9 (1 36) 4 ( ) 5 (1 50) PEI, patients who received cisplatin, etoposide and ifosfamide (PEI); PBSC, peripheral blood stem cell; WBC, white blood cell count; rhg-csf and rhgm- CSF, recombinant human granulocyte and granulocyte macrophage colony-stimulating factor, respectively. Data present median values and ranges. a Significant difference (P <0.05) in comparison with rhgm-csf. phamide anthracycline regimen, on the other hand, appeared to be superior to ID HD cyclophosphamide [6] (Table 7). There was also a difference between the two groups of ifosfamide etoposide-based regimens, favoring ifosfamide etoposide plus or minus Ara-C or VCR over ifosfamide etoposide cisplatin, but at borderline significance (Table 8). Logistic regression analysis The impact of hematopoietic growth factor and chemotherapy regimen, as well as that of age ( 40 versus >40 years), sex, type of malignancy, pretreatment (yes versus no) and the number of relapses (0 5) on the mobilization and collection of CD34+ cells was tested in a logistic regression analysis, including the yield of at least CD34+ cells as the target. In this analysis, in accordance with the data presented above, the use of ifosfamide etoposide-based regimens or other drug combinations and the use of rhg-csf or rhgm-csf independently and significantly (P = and P = 0.030, respectively) influenced the harvest of CD34+ cells. Another significant factor was the number of relapses the patients had before receiving the current salvage treatment (P = 0.011). Discussion Table 5. PBSC mobilization and collection in patients receiving rhg-csf and ifosfamide etoposide ± Ara-C or VCR (A) or cyclophosphamide anthracycline (B) In accordance with other studies [52 54], our results show a high predictive value of CD34+ cell concentration in the peripheral A (n = 50) B (n = 49) P Day of 1st PBSC collection 14 (10 26) 13 (11 16) WBC at 1st PBSC collection (10 3 /µl) 5.9 (0.6 42) 16.3 (4 48.8) at 1st PBSC collection 119 ( ) 87(14 579) blood processed 48 ( ) 31 (4 118) CD34+ cell yield (10 6 /kg body wt) 13 ( ) 7(1 24) Ara-C, cytosine arabinoside; PBSC, peripheral blood stem cell; rhg-csf, recombinant human granulocyte colony-stimulating factor; VCR, vincristine; WBC, white blood cell count. Data present median values and ranges. blood and the yield of these cells in the apheresis product. The number of CD34+ cells collected increased with their increasing level in the peripheral blood, but the collecting efficiency, defined as the proportion of cells that could be collected from each microliter of blood, decreased from a median of 0.36 on the first day of apheresis to a median of 0.31 and 0.24 on the second and third day, respectively. Using such collection efficiency values and the following formula, it is possible to calculate roughly the expected yield of CD34+ cells at a certain concentration of these cells in the PB and to calculate the volume of the blood that should be processed in order to obtain a certain target amount of CD34+ cells/kg body wt (Figure 1): [target yield of CD34+ cells (10 6 ) body wt (kg)] : [CD34+ cell count in the peripheral blood (µl) collection efficiency] = blood volume to be processed (l). The collection efficiency of CD34+ cells, however, may differ considerably in each patient from the median value, probably related to variations in apheresis technique and individually varying drop of CD34+ cell count and additional CD34+ cell mobilization during CD34+ cell harvest [55, 56]. ID HD cyclophosphamide is frequently used to mobilize and collect PBSC [1, 3, 5, 9, 10, 12 14, 16, 17, 32]. Other frequently used regimens are ifosfamide etoposide-based regimens [25 28, 30 35, 37 42] and combinations of CD cyclophosphamide with various drugs, such as anthracyclines [8, 25]. It is therefore of particular interest to evaluate and compare the efficacy of these regimens to define the impact of the type of chemotherapy on the

5 i33 Table 6. PBSC mobilization and collection in patients receiving rhg-csf and ifosfamide etoposide ± Ara-C or VCR (A) or ID HD cyclophosphamide (B) A (n = 50) B (n = 29) P Day of 1st PBSC collection 14 (10 26) 13 (10 21) WBC at 1st PBSC collection (10 3 /µl) at 1st PBSC collection blood processed CD34+ cell yield (10 6 /kg body wt) 5.9 (0.6 42) 13.9 ( ) ( ) 59 (10 459) ( ) 21 (2 161) ( ) 5 (0.6 45) Ara-C, cytosine arabinoside; ID HD, intermediate to high dose; PBSC, peripheral blood stem cell; rhg-csf, recombinant human granulocyte colony-stimulating factor; VCR, vincristine; WBC, white blood cell count. Data present median values and ranges. Table 7. PBSC mobilization and collection in patients receiving rhg-csf and ID HD cyclophosphamide (A) or CD cyclophosphamide anthracycline (B) A (n = 29) B (n = 49) P Day of 1st PBSC collection 13 (10 21) 13 (11 16) WBC at 1st PBSC collection (10 3 /µl) at 1st PBSC collection blood processed CD34+ cell yield (10 6 /kg body wt) 13.9 ( ) 16.3 (4 48.8) (10 459) 87 (14 579) (2 161) 31 (4 118) (0.6 45) 7.3 (1 24) CD, conventional-dose; ID HD, intermediate to high dose; PBSC, peripheral blood stem cell; rhg-csf, recombinant human granulocyte colony-stimulating factor; WBC, white blood cell count. Data present median values and ranges. outcome of PBSC mobilization and collection. The results of our study clearly show a strong relationship between the chemotherapy regimen used and the amount of CD34+ cells that can be collected. Such a relationship has also been observed in some other studies comparing the efficacy of various chemotherapy regimens [8, 13, 16, 25, 32]. In one study, the combination of ifosfamide, etoposide and epirubicin was found to be superior to ID cyclophosphamide [32], and in another study, the addition of etoposide to ID cyclophosphamide improved the results of PBSC mobilization and collection [13]. In our study, ifosfamide etoposide-based regimens, particularly those containing ifosfamide plus etoposide alone or with Ara-C or VCR, appeared to be significantly more effective than ID HD cyclophosphamide or a combination of CD cyclophosphamide and an anthracycline. Ifosfamide etoposide-based regimens are frequently used in patients with malignant lymphoma, testicular cancer, lung cancer and other solid tumors, such as Ewing s sarcoma and alveolar Table 8. PBSC mobilization and collection in patients receiving rhg-csf and ifosfamide etoposide cisplatin (A) or ifosfamide etoposide ± Ara-C or VCR (B) A (n = 20) B (n = 50) P Day of 1st PBSC collection 16 (13 20) 14 (10 26) WBC at 1st PBSC collection (10 3 /µl) at 1st PBSC collection CD34+ cells/ml blood processed CD34+ cell yield (10 6 /kg body wt) 10 (2 33.1) 5.9 (0.6 42) ( ) 119 ( ) (4 210) 48 ( ) ( ) 13 ( ) Ara-C, cytosine arabinoside; PBSC, peripheral blood stem cell; rhg-csf, recombinant human granulocyte colony-stimulating factor; VCR, vincristine; WBC, white blood cell count. Data present median values and ranges. rhabdomyosarcoma, since, beside their efficacy in mobilizing PBSC, they represent highly effective treatments of these diseases [27, 28, 32 37, 39 42, 57, 58]. Such regimens have also been used for mobilizing PBSC in patients with chronic myelogenous leukemia and multiple myeloma [30, 31, 38, 59]. Another important factor determining the yield of PBSC found in our study was the number of relapses that the patients had before they received the current salvage chemotherapy to mobilize CD34+ cells. This finding confirms the results of other studies indicating a negative relationship between the intensity of cytotoxic pre-treatments, particularly prolonged application of alkylating agents, and the outcome of CD34+ cell mobilization and collection [3, 5, 13, 15, 17, 60 63]. PBSC harvest should, therefore, be planned as early as possible in the course of disease of patients who are candidates for autologous PBSC transplantation. As a single agent, rhg-csf has been shown to mobilize more CD34+ cells than rhgm-csf [46 48]. In combination with chemotherapy, however, there are controversial results regarding the potential of the two factors in mobilizing CD34+ cells. While in a small randomized study, the two growth factors showed similar activity [49] in two other randomized studies, including a study with a large number of patients, rhg-csf appeared to be superior to rhgm-csf in mobilizing CD34+ cells [50, 51] and, additionally, in reducing the toxicities of chemotherapy [51]. Our study similarly shows a significant difference between the two growth factors, favoring rhg-csf. This difference was also evident when the effects of rhg-csf and rhgm-csf were compared in a homogenous group of patients with regard to age, gender and chemotherapy regimen. In addition, the type of growth factors used was found to be of independent significance for mobilizing CD34+ cells and achieving the target yield of these cells in logistic regression analysis. In conclusion, the results of the present study show a significant impact of the type of chemotherapy and hematopoietic growth factor on mobilization and yield of PBSC. The results further indicate rhg-csf as a factor with a greater potential of

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