E Aurlien, H Holte, A Pharo, S Kvaløy, E Jakobsen, EB Smeland and G Kvalheim. Summary:

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1 Bone Marrow Transplantation, (1998) 21, Stockton Press All rights reserved /98 $12. Combination chemotherapy with mitoguazon, ifosfamide, MTX, etoposide (MIME) and G-CSF can efficiently mobilize PBPC in patients with Hodgkin s and non-hodgkin s lymphoma E Aurlien, H Holte, A Pharo, S Kvaløy, E Jakobsen, EB Smeland and G Kvalheim University Hospital, The Norwegian Radium Hospital, Oslo, Norway Summary: Many centers use CY and G-CSF to mobilize PBPC. In this study we explored whether a standard chemotherapy regimen consisting of mitoguazon, ifosfamide, MTX and etoposide (MIME) combined with G-CSF was capable of mobilizing PBPC in lymphoma patients. Twelve patients with Hodgkin s disease (HD) and 38 patients with non-hodgkin s lymphoma (NHL) were mobilized with MIME/G-CSF. Most patients were heavily treated with different chemotherapy regimens receiving a median of 11 cycles (range 3 to 2) of chemotherapy prior to mobilization. It was found that the optimal time of PBPC harvest was at days 12 and 13 after initiating the mobilization regimen. The median number of collected CD34 cells per kg body weight was (range ). More than CD34 cells/kg were achieved in 69% of the patients after one apheresis. When additional cycles of apheresis were done, only 6% failed to harvest this number of CD34 cells. There was a statistically significant inverse correlation between the number of prior chemotherapy cycles and CD34 cell yield (P.3). No such association was found between CD34 cell yield and prior radiotherapy. When MIME/G-CSF was compared with Dexa-BEAM/G-CSF, it was found that MIME/G-CSF tended to be more efficient in mobilizing PBPC in spite of being less myelotoxic. All patients transplanted with MIME/G-CSF mobilized PBPC had fast and sustained engraftment. These results demonstrate that an ordinary salvage chemotherapy regimen, such as MIME combined with G-CSF can be successfully used to mobilize PBPC. Keywords: PBPC mobilization; CD34 + cells; combination chemotherapy; MIME; G-CSF; malignant lymphoma High-dose therapy (HDT) with autologous stem cell support is increasingly used to treat selected patients with malignant lymphomas. Several studies have shown that the Correspondence: Dr G Kvalheim, Clin Stem Cell Laboratory, University Hospital, The Norwegian Radium Hospital, Montebello, 31 Oslo, Norway Received 27 September 1997; accepted 4 December 1997 use of PBPC grafts gives a faster reconstitution of neutrophils and platelets compared to BMT. 1 3 In relapsed patients previously treated with chemotherapy, combinations of chemotherapy and growth factors appear to be the most efficient PBPC mobilization regimen. 4 6 Single high-dose CY has been frequently used to mobilize PBPC in combination with G-CSF or GM-CSF. Accumulating evidence using this combination suggests a dose relationship between CY and the yield of PBPC. Unfortunately, this is also associated with increased incidence of severe toxicity. 7 9 In patients with relapsed HD and high-grade NHL, primarily treated with doxorubicin-containing chemotherapy, we use MIME as induction therapy prior to HDT with autologous stem cell support. MIME induces 5 to 6% complete or partial remissions, which is comparable with other salvage regimens. 1,11 The present study was designed to determine the efficacy of MIME combined with G-CSF in PBPC mobilization. The optimal timing of progenitor cell collection, adverse effects of mobilization and collection and the time to hematopoietic reconstitution after HDT were determined. Our data show that among the 5 lymphoma patients mobilized with the MIME/G-CSF combination, only 6% failed to harvest CD34 + cells/kg. No severe adverse events were observed prior to or during the PBPC collections, and all patients reconstituted with MIME/G-CSF primed products had a fast and durable hematopoietic engraftment. Patients and methods Patients studied From April 1994 to January 1997, 6 patients with malignant lymphoma were selected for HDT with PBPC support. Three patients were mobilized with CY/G-CSF and are not included in the further analyses. Seven HD patients were mobilized with Dexa-BEAM/G-CSF. Of the remaining 5 patients mobilized with MIME/G-CSF, 12 had HD and 38 NHL. Seventy percent of these were male and the median age was 4 years (range 14 6). Prior to mobilization of PBPC, most patients were heavily treated with different chemotherapy regimens receiving a median of 11 chemotherapy cycles (range 3 2). Nineteen (38%) had received localized radiotherapy, including nine treated with extended field radiation.

2 874 Enumeration of CD34 + cells and hematopoietic colonyforming cells Total nucleated cells (TNC) in the PBPC products were counted employing a Cell dyn 35 cell counter (Abbott Laboratories, Irvine, CA, USA). Enumeration of CD34 + cells in blood and in the apheresis product were determined by use of a FACSort flow cytometer (Becton Dickinson, San Jose, CA, USA), 12 using a Nordic standardization method, as described previously. 13 Colony-forming cell (CFU-c) assay was performed employing a standardized method with media containing appropriate amounts of growth factors (Methocult H4433; Stem Cell Technologies, Vancouver, Canada). 14 PBPC mobilization and collection The MIME chemotherapy regimen used for mobilization consists of i.v. infusions of mitoguazon 5 mg/m 2 at day 1, ifosfamide 1 mg/m 2 daily on 5 consecutive days with mesna 2 mg/m 2 three times a day, MTX 3 mg/m 2 at day 3 and etoposide 1 mg/m 2 daily from day 1 to 3. Two days after the chemotherapy, at day 7, s.c. G-CSF 5 g/kg was administered, and continued daily until an adequate number of CD34 + cells was collected. Dexa-BEAM 15 (dexamethasone, BCNU, etoposide, ara-c and melphalan), was combined with G-CSF 5 g/kg s.c. from day 8. When increasing levels of CD34 + cells appeared in the blood, leukapheresis was performed with a CS 3 Fenwall Cell Separator (Baxter, Deerfield, IL, USA). Ten liters of blood were processed for 1 to 3 days until the required yield of CD34 + cells was reached. High-dose therapy Of the 5 patients mobilized with MIME/G-CSF, 4 were treated with HDT consisting of either TBI followed by CY, (TBI/CY) 16 (n = 14), BEAM 1 (n = 25), or BEAC 16 (n = 1). Stem cells were given as PBPC only in 35 patients, one received BM, and four a combination of BM and PBPC. Time to neutrophil recovery is defined as the time from PBPC infusion to the first day when sustained counts of /l was reached. Platelet recovery is defined as the time until a sustained platelet count was /l, without platelet transfusions. Statistical analysis of data For statistical analysis we employed SPSS 6.1 for Windows (SPSS Inc, Chicago, IL, USA). Values given are mean, median and range. For comparing groups of patients we employed linear regression analysis and two-sample t-test. Results Time for collection of PBPC The number of mobilized CD34 + cells and the optimal time for collection of PBPC might differ when single drug or combination chemotherapy regimen are used together with G-CSF. For the lymphoma patients mobilized with MIME/G-CSF, frequent measurements of CD34 + cells in blood were done during recovery after chemotherapy. We observed a wide range of detectable levels of CD34 + cells from day 11 until day 15 after initiating the mobilization regimen. As can be seen in Table 1, most patients had peak CD34 + cell levels at day 13 and 14. Patients that experienced late peaks of CD34 + cells had lower levels than those who reached their peak before day 14. Appropriate levels of CD34 + cells to start leukapheresis were, in the majority of patients, present before the peak levels appeared. Consequently, in our MIME/G-CSF mobilized patients, 11 started harvesting at day 11 or 12, 34 at day 13, and four at day 14. Mobilization efficacy and side-effects The median number of collected CD34 + cells/kg in the MIME/G-CSF mobilized patients was (range.5 to 26.2). More than CD34 + cells/kg were achieved in 69% of the patients after one apheresis. Previously it has been indicated that infusion of CD34 + cells/kg after HDT is associated with predictable and early platelet engraftment. 17 This proposed number of CD34 + cells could be collected after one apheresis in 55% of the patients and in 8% if a second apheresis was added. Six patients out of 5 developed neutropenic fever, and no platelet transfusions were required. Only three patients mobilized poorly with MIME/G-CSF, and required BM harvest in addition to PBPC. Two of them were diagnosed with herpes zoster infection at the time of leukapheresis, and one developed pericarditis. Efficacy of mobilization with MIME/G-CSF related to lymphoma subgroup, previous radio- and chemotherapy Before PBPC mobilization, both HD and NHL patients had received a similar number of chemotherapy cycles. Patients with HD had more extended fields of radiotherapy. As can be seen in Figure 1a, we did not find any significant difference in mobilization capability the first day of apheresis between patients with HD and NHL patients (P =.67). Increasing numbers of chemotherapy cycles contributed to a significant inverse relation to the number of CD34 + cells harvested the first day of apheresis (P =.3). Statistically it was found that one additional chemotherapy cycle reduced the first day harvest with CD34 + cells/kg. These results also persisted when prior radio- Table 1 Days after initiating the mobilization treatment, and peak levels of CD34 + cells in blood No. of patients Peak levels of CD34 + cells 1 6 /l At day Median (range) ( ) ( ) ( ) ( )

3 CD34+ cells 16/kg first day apheresis P =.67 P =.3 P =.37 HD NHL 11 cycles 12 cycles none involved field wide field (a) Lymphoma subgroup (b) Previous chemotherapy (c) Radiotherapy prior to mobilization 875 Figure 1 Yield of CD34 + cells/kg after first day of apheresis in individual patients mobilized with MIME and G-CSF. Median and P values are included. Mobilizing efficacy is compared between (a) patients with Hodgkin s disease (HD) and non-hodgkin lymphoma (NHL), and related to (b) number of chemotherapy cycles before mobilization and (c) previous radiotherapy given as none, involved or wide field. therapy was taken into account (P =.2). By inspection of the data, it was found that patients given 12 cycles of chemotherapy obtained a poorer yield of CD34 + cells, as visualised in Figure 1b. In Figure 1c the first day of leukapheresis collections of CD34 + cells/kg were compared in patients given none, involved field or wide field radiotherapy. Even if these data indicate a negative association between radiotherapy and the yield of CD34 + cells, this was not found to be statistically significant (P =.37). Mobilization of PBPC with Dexa-BEAM or MIME plus G-CSF in patients with HD In 19 patients with HD, the mobilization efficacy was compared, employing either Dexa-BEAM/G-CSF or MIME/G- CSF (Table 2). The MIME/G-CSF mobilized patients had no preferences in variables, possibly influencing the mobilization ability. Three of seven Dexa-BEAM/G-CSF mobilized patients achieved CD34 + cells/kg, while 11 out of 12 MIME/G-CSF patients mobilized more than this number of CD34 + cells. The median nadir of leukocytes and platelets following MIME/G-CSF was significantly higher compared to Dexa-BEAM/G-CSF (P.1). Consequently, the number of neutropenic and thrombocytopenic events was considerably reduced. To explore the differences among these mobilizing regimens further we studied one patient mobilized sequentially with Dexa-BEAM/G-CSF and MIME/G-CSF (Figure 2). A HD stage IVA patient, previously treated with 1 chemotherapy cycles and mantle field radiation, was mobilized up front with Dexa-BEAM/G-CSF due to a relapse 2 months after previous treatment. At the time of mobilization, he had bone and marrow involvement, but no B symptoms. During the recovery phase a significant myelotoxicity was observed, giving neutropenic fever and severe thrombocytopenia lasting for 5 days. Since the highest level of CD34 + cells detected in the blood was too low to expect PBPC yield of minimum CD34 + cells/kg, the patient was not harvested. Thirty-one days after initiating the Dexa- BEAM regimen, the patient was mobilized with MIME combined with G-CSF. In one single apheresis at day 13, a yield of CD34 + cells/kg was achieved. During both mobilization cycles the patient was given s.c. G- CSF 1 g/kg/day. Engraftment characteristics after reinfusion of PBPC in MIME/G-CSF mobilized patients Following HDT, 35 patients received PBPC mobilized with MIME/G-CSF. They were reinfused with a median of CD34 + cells/kg (range 2.2 to 2.) and only two patients were given G-CSF post-transplant. Neutrophil recovery was achieved after a median number of 12 days (range 8 24). Platelet independence was reached at day 11 (median). One patient died in multi-organ failure 43 days after reinfusion of PBPC. She required platelet transfusions until she died. The remaining patients recovered platelets within the range of 7 to 27 days. There was a positive relation between number of CD34 + cells reinfused and engraftment of neutrophils (P =.15) and platelets (P =.14). We observed a borderline association between the number of CFUs reinfused and time to engraft neutrophils (P =.52), but no relation to platelet reconstitution (P =.135). In contrast, an association was found between platelet engraftment and number of chemotherapy cycles prior to mobilization (P =.26). When the number of CD34 + cells/kg reinfused was taken into account in a multiple linear regression analysis, the association persisted, although it was not formally statistically significant (P =.53). Discussion The MIME regimen is widely used in Scandinavia as second-line chemotherapy for patients with relapsed HD and high-grade NHL, who previously have received doxorubicin-containing regimens. In the present study, we explored the use of MIME combined with G-CSF for the purpose of mobilizing PBPC. The rational for such an approach is two-fold. First to obtain an in vivo tumor cell

4 876 Table 2 Comparison between MIME/G-CSF and Dexa-BEAM/G- CSF as PBPC mobilizing regimens in patients with HD. Patients characteristics, mobilization efficacy and side-effects MIME/G-CSF Dexa-BEAM/ G-CSF Total No. of patients 12 7 Disease stage at relapse (n) I 1 II 4 4 III 3 2 IV 4 1 B symptoms at relapse 8 5 BM involvement at relapse 3 Disease state at mobilization (n) 2.CR PR PR PR 1 Prior radiotherapy given 7 3 Prior chemotherapy cycles, median (range) (7 13) (9 14) Previous MIME or Dexa-BEAM 4 2 cycles, median (range) (3 6) (1 3) Days from last chemo cycle to start mobilization, median 21 3 (range) (21 28) (21 36) Nadir leukocytes 1 9 /l median (range) (.4 6.8) (.3) Nadir platelets 1 9 /l median (range) (31 161) (7 24) Neutropenic fever 1 5 Platelet transfusions 4 WBC 1 9 /l the first day of apheresis, median (range) ( ) ( ) No. of aphereses 2 I 4 2 II 6 2 III 2 1 CD34 + cells 1 6 /kg median (range) (.5 1.8) (. 24.) treatment effect of MIME, and secondly to use the same drugs in combination with G-CSF for stem cell mobilization. In this study, including 5 pre-treated patients, a predictable increasing level of CD34 + cells appeared in the blood at days 12 and 13 after MIME/G-CSF. The median number of collected CD34 + cells was for the MIME/G-CSF mobilized patients /kg. A yield of more than CD34 + cells/kg were achieved in 69% of the patients after one apheresis, while CD34 + cells/kg were harvested after one apheresis in 55% of the patients. The method most commonly used for PBPC mobilization is a single dose of CY 1.5 g/m 2 plus G-CSF. When such a regimen was employed by Watts et al 17 in a similar cohort of lymphoma patients, only 28% obtained CD34 + cells/kg after one apheresis while 5% achieved this number after two aphereses. Although higher doses of CY such as 4 to 7 g/m 2 with G-CSF or GM-CSF give a higher yield of CD34 + cells in the PBPC products, the incidence of side-effects increases considerably. 9 The combination of ifosfamide plus etoposide and G- CSF was used by Baars et al 18 to mobilize 41 patients with malignant lymphoma or solid tumors. After 1 day of collection, 51% of their patients mobilized CD34 + cells/kg. In another study on relapsed malignant lymphoma patients 19 the combination of ifosfamide, etoposide and epirubicin plus G-CSF collected per leukapheresis a median of CD34 + cells/kg (range.1 21). Among our MIME/G-CSF patients the median yield per leukapheresis was CD34 + cells/kg (range ). It is not known which of the individual drugs in the MIME combination contributes to the efficient mobilization of PBPC. Since the doses of ifosfamide and etoposide successfully used in a previous study 19 are similar to those used in the MIME regimen, we have reason to believe that these two drugs might be the major contributors to the efficient MIME/G-CSF PBPC mobilization. It has been claimed that patients with HD mobilize PBPC less efficiently than other lymphomas. 2 Both our HD and NHL patients had similar yields of CD34 + cells on the first day of apheresis. The amount of previous chemotherapy and irradiation have been reported to negatively influence the yield of Leukocytes 19/l Nadir leukocytes Platelets 19/l Nadir platelets Cd34+ cells 16/l Days from start mobilization therapy CD34+ cells in blood Figure 2 A Hodgkin s patient was mobilized sequentially with dexa-beam/g-csf (white bars) and MIME/G-CSF (black bars). Left bars show nadir values of leukocytes and platelets, employing the different mobilizing regimens. Right bars demonstrate the differences in CD34 + cells measured in blood during 2 and 3 consecutive days.

5 mobilized CD34 + cells. 5,17,2,21 In keeping with some reports and in contrast to others, 2 22 we observed that the number of chemotherapy cycles prior to MIME/G-CSF mobilization significantly influenced the yield of CD34 + cells. No such association could be found between previous irradiation and yield of CD34 + cells, although a trend was seen. The Dexa-BEAM/G-CSF combination can be used to mobilize patients with relapsed HD. 15 When this regimen was compared with MIME/G-CSF in the same type of HD patients it was found that Dexa-BEAM/G-CSF tended to mobilize less CD34 + cells. Previously it has been suggested that when combinations of chemotherapy and G-CSF are used as mobilizing regimens, a direct correlation between myelosuppression and number of mobilized CD34 + cells exists. 9 For unknown reasons this was not the case when MIME/G-CSF was used since the median nadir value of leukocytes was only /l and platelets /l. As a result of this, none of the 5 patients needed platelet transfusions and only six patients had neutropenic fever. In contrast, among Dexa-BEAM/G-CSF-mobilized patients, who had profound myelosuppression, five of seven patients had neutropenic fever and four required platelet transfusions. All of the MIME/G-CSF-mobilized patients experienced fast engraftment, with neutrophil and platelet recoveries at median days 12 and 11, respectively. The hematopoietic reconstitution was independent of the different HDT regimens. The number of chemotherapy cycles, but not irradiation, prior to mobilization influenced the time to platelet engraftment. The reason for this observation is not clear. In accordance with earlier reports, there was a significant positive association between the number of re-infused CD34 + cells and engraftment of neutrophils (P =.15) and platelets (P =.14). Our experience in lymphoma patients with enrichment of CD34 + cells employing Isolex 3I show a purity of CD34 + cells of 96 to 99%, but with a yield of only 4 to 5%. 23,24 Consequently, achieving purified CD34 + cells/kg requires that the starting product contains at least CD34 + cells/kg. Seventy-two percent of our patients mobilized with MIME/G-CSF succeeded in yielding this number of CD34 + cells/kg. Recently, in three patients with HD, we employed MIME and G-CSF 1 g/kg instead of 5 g/kg. All three patients mobilized a high yield of CD34 + cells (data not shown). If these results can be reproduced in a larger number of patients, this approach may further increase the efficacy of the MIME/G-CSF mobilizing regimen. Based on our data, we conclude that MIME/G-CSF is an efficient mobilization regimen giving less myelotoxicity than comparable regimens. Since this also reduces the frequency of neutropenic fever and platelet transfusions such a regimen should be favorable in pre-treated lymphoma patients. Acknowledgements This study is supported by The Norwegian Cancer Society. The authors are indebted to Dr E Skovlund for valuable statistical help and discussion. We thank E Lenschow, K Ugland, E Gilen and I Hervik for outstanding technical assistance, and the nursing staff at the transplantation unit. References 1 Schmitz N, Linch DC, Dreger P et al. Randomised trial of filgrastim peripheral blood progenitor cell transplantation vs autologous bone marrow transplantation in lymphoma patients. Lancet 1996; 347: To LB, Roberts MM, Haylock DN et al. Comparison of haematological recovery times and supportive care requirements of autologous recovery phase peripheral blood stem cell transplants, autologous bone marrow transplants and allogeneic bone marrow transplants. 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