Hemopoietic Progenitor Cell Mobilization and Harvest Following an Intensive Chemotherapy Debulking in Indolent Lymphoma Patients

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1 Hemopoietic Progenitor Cell Mobilization and Harvest Following an Intensive Chemotherapy Debulking in Indolent Lymphoma Patients CORRADO TARELLA, a FRANCESCO ZALLIO, a DANIELE CARACCIOLO, a CRISTINA CHERASCO, a PAOLA BONDESAN, a PAOLO GAVAROTTI, a PAOLO CORRADINI, a VALTER TASSI, b ALESSANDRO PILERI a a Dipartimento di Medicina e Oncologia Sperimentale, Divisione Universitaria di Ematologia, Torino, Italy; b Banca del Sangue, Fondazione G. Strumia. Az. Ospedaliera S. Giovanni Battista, Torino, Italy Key Words. PBPC Mobilization Chemotherapy Tumor contamination Indolent lymphoma ABSTRACT An in vivo purging with intensive debulking chemotherapy prior to peripheral blood progenitor cell (PBPC) collection may reduce the risk of tumor contamination of the harvest products; however, it is usually associated with a marked reduction in PBPC mobilization. These issues have been considered while designing an adapted version of the high-dose sequential regimen for patients with lymphoid malignancies and bone marrow involvement. To reduce tumor contamination risks, PBPC collection was postponed to the end of the high-dose phase; however, in order to enhance progenitor cell mobilization, a chemotherapy-free lag period was introduced prior to the final mobilizing course. Thirty-nine patients (median age 47 years, range 26-62) with previously untreated indolent lymphoma entered this pilot study; all had advanced-stage disease, and 29 had overt marrow involvement. Sufficient numbers of PBPC to perform autograft with safety were harvested in 34 patients, with a median of 3 (range 2-5) leukaphereses. A median of (range 2-51) CD34 + /kg and (range ) colony forming units-granulocyte/macrophage/kg were collected per patient. In univariate analysis, the duration of the chemotherapy-free interval prior to the final mobilizing course, i.e. > or <65 days, was the most significant variable influencing progenitor mobilization. These data suggest that extensive in vivo tumor debulking is feasible provided that a sufficient chemotherapy-free period preceding the mobilizing course is allowed in order to allow a full recovery of marrow functions. Stem Cells 1999;17:55-61 INTRODUCTION The use of intensive chemotherapy with bone marrow (BM) autograft has been recently proposed as a novel therapeutic option for younger indolent lymphoma patients [1]. Promising results have been reported in both chronic lymphocytic leukemia and follicular lymphomas [2-4]. The use of mobilized peripheral blood progenitor cells (PBPC) may further widen the use of this approach [5-7]. However, marrow involvement is almost the rule in indolent lymphomas. This may hamper the applicability of PBPC autograft. Indeed, marrow involvement may preclude an adequate PBPC mobilization and harvest [8, 9]. In addition, PBPC harvest in patients with extensive marrow involvement implies an increased risk of tumor contamination in the harvested material, thus reducing the potential advantages of autograft-based programs [10]. Tumor debulking prior to mobilization could imply better conditions for optimal PBPC harvest. However, previous cytotoxic treatments have been shown to adversely influence progenitor mobilization [9, 11-14]. In particular, mobilization is negatively affected by a preceding cytotoxic treatment delivered a short time before [11, 15, 16]. These observations may preclude the possibility of scheduling PBPC collection following extensive tumor debulking with repeated chemotherapy courses. Acting on these premises, we designed a modified version of the high-dose sequential (HDS) chemotherapy regimen, a scheme successfully employed in the treatment of aggressive non-hodgkin s lymphoma [7, 17, 18]. Modifications were aimed at obtaining a more intense in vivo purging by intensifying tumor debulking prior to PBPC collection. Recently, we Correspondence: Dr. C. Tarella, Divisione Universitaria di Ematologia, Az. Ospedaliera S. Giovanni Battista, Via Genova 3, Torino, Italy. Accepted for publication October 27, AlphaMed Press /99/$5.00/0 STEM CELLS 1999;17:55-61

2 56 PBPC Harvest Following Tumor Debulking reported that such an approach is able to reduce the number of residual neoplastic cells below the detection level of the polymerase chain reaction (PCR) assay both in vivo and in the harvest products of many patients with follicular lymphoma [19]. Feasibility of PBPC collection in indolent lymphoma patients following such a prolonged treatment is described here. The schedule was employed in 39 previously untreated patients with slowly growing lymphomas, including lymphocytic, follicular, and mantle cell subtypes. The results show that a good PBPC collection is still feasible following an intense debulking, provided that an appropriate chemotherapy-free interval is allowed prior to the final mobilizing course. PATIENTS AND METHODS Patient Characteristics Thirty-nine patients with indolent lymphoma were treated with the intensified HDS (i-hds) protocol. Their main clinical features are summarized in Table 1. Eligibility criteria for entering the study included: age between 16 and 60 years; no other associated neoplasia; no previous cytotoxic or radiation therapy; negative tests for HCV-HBsAg-HIV; absence of meningeal involvement; and normal cardiac, renal, and pulmonary function. All patients gave written informed consent for the proposed treatment program. Treatment Schedule The schedule was developed by modifying the previously described HDS chemotherapy regimen [17, 18]. The original HDS regimen includes the sequential administration of: A) high-dose (hd) cyclophosphamide (CY) given at 7 g/m 2 i.v., with PBPC harvest at hemopoietic recovery; B) methotrexate (8 g/m 2 ) plus vincristine (2 mg i.v.) approximately at day 16; C) hd-etoposide (VP16) at 2 g/m 2, approximately at day 23, and D) submyeloablative treatment with PBPC autograft approximately Table 1. Main clinical features of the 39 lymphoma patients treated with the intensified HDS regimen Parameter n (%) Age, median (range) years 47 (30-62) Sex M/F 23/16 Histology*: Low-grade 21 (54) Intermediate-grade 14 (36) Transformed 4 (10) Stage III/IV 37 (95) BM involvement 29 (86) *Histology was defined according to the Working Formulation. between days 48 and 52. The main modification consisted of intensive debulking prior to the hd-phase with two complete APO courses at full dose [20]. Patients not in complete remission (CR) after APO received two additional DHAP courses [21]. In the hd-phase, the CY/VP16 sequence was inverted so that PBPC harvest could be scheduled at the end of the hd-scheme. In addition, a chemotherapy-free interval of 30 days was introduced prior to hd-cy in order to allow adequate marrow repopulation and optimal progenitor cell mobilization [15]. In the chemotherapy-free interval, a total of three hd-dexamethasone courses (dexamethasone at 40 mg/d for four consecutive days) were administered every 10 days. G- CSF (Filgrastim) was given at 5 µg/kg/day following CY, VP16, and after autograft, until myelopoietic recovery. The combined administration of hd-mitoxantrone and hd- L-PAM was used as a conditioning regimen in all patients [22]. Mitoxantrone was given i.v. at 60 mg/m 2 in three divided doses of 1 h each every 2 h on day -5; L-PAM was given i.v. at 180 mg/m 2 in three divided doses of 30 min each every 2 h on day -2; PBPC were reinfused two days later. Radiotherapy was given on bulky sites approximately two months after autograft. The whole i-hds is summarized in Table 2. Collection and Evaluation of Hemopoietic Progenitors PBPC were mobilized and collected after hd-cy. To predict the number and timing of leukaphereses, circulating CD34 + cells along with cell blood counts were evaluated daily starting from day +9 following chemotherapy administration until completion of harvesting procedures. CD34 + cell Table 2. Schedule of the intensified high-dose sequential (HDS) regimen APO-1 d 1 APO-2 d 43 DHAP-1 d 76 DHAP-2 d 98 Etoposide 2 g/m 2 + G-CSF for days d 1 Methotrexate 8 g/m 2 + VCR 2 mg + folinic rescue d 16 DEX days: 26, 36, 46 Cyclophosphamide 7 g/m 2 d 60 + G-CSF for days PBPC harvest d Mitoxantrone 60 mg/m 2 d 94 + L-PAM 180 mg/m 2 d 97 PBPC autograft d 99 G-CSF was given after etoposide and cyclophosphamide at 5 µg/kg/d. Dexamethazone was given at 40 mg/d for four consecutive days, at the given intervals. d = approximate day of drug delivery.

3 Tarella, Zallio, Caracciolo et al. 57 evaluation was carried out by flow cytometric direct immunofluorescence on whole blood samples, according to published procedures [23]. A phycoerythrin-conjugated anti-cd34 monoclonal antibody was used (anti-hpca-2, Becton Dickinson; Lincoln Park, NJ), as described [24]. The number of circulating CD34 + cells per µl of blood was obtained by multiplying the percentage of CD34 + cells by the number of leukocytes in 1 µl of blood. Circulating myeloid progenitors (CFU-GM) were evaluated daily as well, from day +9. The in vitro colonyforming assay was performed by plating total peripheral blood leukocytes obtained after RBC sedimentation in the presence of 33% Emagel (Behring; Marburg, Germany), as described elsewhere [14, 24]. Using this assay, a median value of 146 colony forming units-granulocyte/macrophage (CFU-GM)/ml was obtained in unstimulated PB from normal donors. Peripheral blood buffy-coat cells were collected when the WBC count was at least 1,000/µl and PB CD34 + cells >10/µl. Leukaphereses were performed using continuous-flow blood cell separators (Cobe-Spectra or Fresenius), and 4.5 to 13 blood liters (median 8.9 l) were processed in each procedure. PBPC estimation in leukapheresis product was carried out before cryopreservation by evaluating both CD34 + cells and CFU-GM, as detailed above. The CFU-GM colony assay was performed by plating in semisolid medium 1, 10 and 100 µl of the buffy coat obtained from leukapheresis cell suspension without any additional cell separation. This prevented any progenitor cell enrichment due to cell separation. The total number of collected CD34 + cells ( 10 6 /kg) and CFU-GM ( 10 4 /kg) was determined by multiplying their frequency per ml by the total volume of cryopreserved cell suspension and dividing by body weight. Values of CFU-GM/kg were taken as the minimal required dose to enroll patients in the autografting program with PBPC only. Supportive Care The entire pretransplant program was carried out in ordinary, nonprotected rooms; for the autograft phase, all patients were admitted to a dedicated inpatient bone marrow transplant unit. During the pancytopenic periods following hd-cytotoxic drug administration and autograft, patients were managed with a common protocol for prophylaxis, including oral ciprofloxacin (500 mg bid) + fluconazole (150 mg) + i.v. acyclovir (250 mg tid); in case of fever >38 C, blood culture and chest x-ray were performed, then patients were empirically treated with i.v. mezlocillin + amikacin + vancomicin; if the fever was of undetermined origin and persisted after h, mezlocillin was replaced by imipenem; i.v. amphotericin was added in a few cases with fever persisting for additional h. Antibiotics were continued until the temperature reverted to normal values for at least two consecutive days along with the return of the ANC to >500/µl. Irradiated, leukocyte-filtered, single-donor platelet concentrates or, less frequently, multiple-donor irradiated platelet concentrates were given when platelet count was less than 20,000/µl; irradiated, leukocyte-filtered packed RBCs were given if hemoglobin was less than 8 g/dl. Statistical Analysis A number of clinical variables potentially influencing PBPC mobilization capacity were considered. The nonparametric Mann-Whitney test was applied to the univariate analysis, and a significant difference was chosen with p level less than RESULTS One patient died of cerebral hemorrhage after completion of the APO courses; he had been receiving warfarin for previous thrombophlebitis, and his hematologic parameters were normal at the time of the hemorrhagic accident. The remaining 38 patients completed the hd-phase and could be evaluated for progenitor mobilization following the final hd-cy and G-CSF. The extent of progenitor mobilization, assessed as peak values of circulating progenitors, is shown in Figure 1. Maximal mobilization occurred at day +14 (range 10-20), with median peak values of circulating CFU- GM, and CD34 + cells of 5,576/ml (range ,440), and 80.3/µl (range ), respectively. Among 38 evaluable patients, 34 (89%) displayed detectable levels of circulating progenitors and underwent PBPC harvest. Adequate numbers of PBPC could be collected through two to five leukapheresis procedures, as detailed in Table 3. A number of clinical variables potentially influencing PBPC mobilization capacity were evaluated. As shown in CFU-GM/ml 30,000 20,000 10,000 0 Figure 1. Circulating progenitor peak values following hdcyclophosphamide and G-CSF delivered after a chemotherapyfree interval. Values of circulating CD34 + cells and CFU-GM recorded in 38 patients during maximal mobilization are plotted as box plots CD34/µl

4 58 PBPC Harvest Following Tumor Debulking Table 4, in univariate analysis, the time elapsed between the cytotoxic mobilizing course and the preceding cytotoxic drug administration was the most significant variant influencing progenitor mobilization; age and performance status at protocol entry also had a significant impact. All four patients displaying no progenitor mobilization had persistent marrow involvement (>30% of marrow infiltration by lymphoma cells) at a biopsy performed immediately prior to hd- CY; however, other patients with persisting marrow involvement had successful mobilization. Thus, marrow involvement was not shown to be an independent factor with significant influence on progenitor mobilization. Table 3. Numbers of harvested progenitor cells in 34 out of 38 indolent lymphoma patients who completed the intensified-hds regimen Median Range n (%) of patients with mobilization* 34 (89%) n of leukaphereses/patient 3 (2-5) Harvested CD34 + cells 10 6 /kg/leukapheresis 3 ( ) Total CD34 + cells 10 6 /kg/patient 14.8 (2-51) Harvested CFU-GM 10 4 /kg/leukapheresis 32.6 ( ) Total CFU-GM 10 4 /kg/patient 100 ( ) *Only 34 of 38 patients displayed signs of even modest mobilization and underwent PBPC harvest. Overall, the whole treatment was well tolerated. There was one toxic death occurring before the start of the hdphase and referrable to warfarin therapy (see above). Hematologic toxicity following the hd-drugs and autograft was superimposable to the one described in previous papers dealing with the original HDS [7, 25]. Extrahematologic toxicity was acceptable. Main complications were fever during the neutropenic phases and oral mucositis. There were three severe infectious complications. One of the first patients entering the study protocol had Pneumocystis carinii pneumonia during the APO courses; since then, cotrimoxazole prophylaxis was introduced and no pneumocystis pneumonia was observed anymore. One patient had pleural empyema that required percutaneous drainage; a microbiological culture was positive for an E. coli, and a pleural infection was successfully treated with systemic antibiotics. A third patient had pneumonia with no positive isolates; however, it resolved after wide-spectrum antibiotic therapy. With the exception of the fatal cerebral hemorrhage, all other complications were reversible. DISCUSSION The HDS regimen has been proposed as a novel treatment approach for chemosensitive tumors [17]. The scheme includes a final autograft with PBPC collected after the initial hd-cy [18]. The use of progenitor cells mobilized and collected in large quantities following a single chemotherapy course may be associated with an increased risk of reinfusion Table 4. Clinical variables potentially influencing PBPC mobilization in 38 evaluable patients treated with HDS Parameter* n CFU-GM/ml p*** peak value** Histology Follicular 17 4,491 Not follicular 21 7, BM involvement at onset Yes 29 5,874 No 9 3, LDH at onset < ,582 > , Severe infection during HDS Yes 4 7,632 No 34 5, Fever during mobilization Yes 7 4,745 No 31 5, BM involvement at the time of mobilization Yes 12 8,522 No 26 5, Interval > 66 days 23 14,784 < 66 days 15 3, *** A univariate analysis of characteristics potentially affecting progenitor cell mobilization was carried out in the 38 patients who received the intensified HDS. *** Mobilization capacity was calculated as peak value of circulating CFU-GM/ml. *** The Mann-Whitney test was used.

5 Tarella, Zallio, Caracciolo et al. 59 of tumor-contaminated material [10, 26-28]. In fact, HDS has been specifically designed for neoplasms with low risks of tumor reinfusion, such as breast cancer or high-grade non- Hodgkin s lymphoma (NHL) lacking BM involvement. In these particular forms, HDS efficacy has been recently documented [25, 29]. Less is known on the potential applicability of HDS in lymphomas presenting with overt marrow involvement. These latter forms would probably benefit by a prolonged tumor debulking treatment prior to mobilization. HDS is characterized by the sequential administration of high-dose cytotoxic drugs. In a previous study, we showed a marked reduction of PBPC mobilization after the second hdcytotoxic course as compared with the first one [15]. This finding is in line with other reports showing a progressive decrease in the mobilization capacity as long as repeated chemotherapy courses are delivered [16, 30]. In addition, we suggested that a delay between cytotoxic courses might somehow restore the mobilization capacity [15]. Our preliminary observation is here adequately documented in a group of 39 patients receiving HDS as first-line therapy. They had indolent lymphoma and were thus at risk for tumor cell reinfusion during autograft. For these reasons, they all received an adapted version of HDS, where intensive debulking was included in order to start the high-dose phase with a reduced tumor burden. Moreover, in this modified version, PBPC harvest is scheduled at the end of a sequence of hd-cytotoxic courses with the aim of achieving a more extensive in vivo tumor debulking. In designing the modified HDS, CY was postponed until the end of the hd-sequence and was preceded by a prolonged chemotherapy-free interval. The tolerability of the modified scheme was overimposable to that observed using the original HDS scheme. A reduced PBPC mobilization was observed following the modified HDS compared with that reported for patients receiving the original HDS [14]. The median peak value of circulating CD34 + cells during mobilization in the original HDS-treated patients has been calculated in the order of 200 to 300 /µl. By contrast, a median peak value of 80 CD34 + /µl was recorded in patients receiving the modified HDS. As a consequence, an increased number of leukapheresis procedures, three per patient as a median, was required for adequate PBPC collections; a single procedure is usually sufficient in the original HDS. Nevertheless, the schedule proved to be efficient since enough PBPC were collected in 34 of 39 patients to perform autograft with safety, and more than CD34 + /kg were harvested in 32 patients. Most patients were in CR at the time scheduled for PBPC collection. This might have coincided with the successful mobilization observed in the majority of patients. In fact, all four of the patients with absent mobilization had BM involvement prior to hd-cy. Thus, although BM involvement did not turn out to be an independent factor with significant adverse influence on PBPC mobilization, reduction of tumor infiltration within the marrow environment may improve the chances of successful mobilization. In addition, the achievement of CR may imply PBPC collections with low or even absent tumor contamination. Indeed, we have recently monitored by PCR analysis the residual tumor contamination in leukapheresis products from follicular and mantle-cell lymphoma patients, and we found that, at least in the follicular subtype, tumor cells are often molecularly undetectable following the intensified HDS schedule [19]. In cases where collections were positive for molecularly detectable residual disease, despite prolonged and intensive treatment, an in vitro purging could be considered. Autograft with PCR-negative material has been shown to be associated with a prolonged survival without disease progression in indolent lymphoma [31]. This has contributed to the increasing interest in the inclusion of in vitro purging procedures in highdose programs with BM or PBPC autograft [2, 32-34]. A reduced tumor contamination and the availability of adequate quantities of hemopoietic progenitors are essential for a safe and successful program completion. In regard to this, our approach seems particularly suitable since it provides extensive tumor reduction in addition to the possibility of collecting sufficient numbers of PBPC to allow for in vitro manipulation. We and others have already shown a reduced mobilization following sequential chemotherapy courses given at tightly spaced intervals [15, 16]. Here, we document that normal marrow functions, including mobilization capacity, can be restored if a given chemotherapy-free interval is allowed prior to the last mobilizing course. Also, it is reasonable to think that harvests might be cleaner if performed after more chemotherapy, due to a more pronounced in vivo purging effect. We propose a period of at least 65 days to guarantee sufficient mobilization. In fact, in univariate analysis, an interval of 65 or more days was associated with the highest progenitor mobilization. During this lag period, only dexamethazone and no cytotoxic drugs were delivered. Such a prolonged chemotherapy-free period could be risky in rapidly growing neoplasms. However, the scheme was specifically designed for indolent lymphomas, i.e., tumors with slow cell growth. No tumor progression was observed in the chemotherapy-free period. Thus, this treatment modality might be preferentially considered for all those chemosensitive neoplasms characterized by a slow growth rate. For neoplasms with more aggressive features, marrow-sparing drugs other than dexamethasone should be included in this phase in order to achieve an effective tumor control. For this purpose, the recently developed anti-cd20 humanized monoclonal antibody could be particularly suitable, since it displays effective antitumor activity without affecting the myelopoietic system [35].

6 60 PBPC Harvest Following Tumor Debulking The risk of tumor contamination in the graft material is of concern not only in hematological neoplasms but also in several other cancers [26-28]. In recent years, a major issue in the field of autograft has been the search for adequate tools to minimize the risk of residual tumor cell reinfusion during autograft. Thus, our schedule, originally designed for indolent lymphomas, might be suitable for other malignancies. The whole program proved to be tolerable, with an overall toxicity analogous to that observed with the original HDS. Recent reports have shown the efficacy of the original HDS both in high-grade NHL as well as in breast cancer [25, 29]. Our intensified and modified HDS version could be applied to breast cancer as well as to other chemosensitive solid tumors, representing an effective alternative to the original HDS for patients at increased risk of marrow involvement by neoplastic cells. ACKNOWLEDGMENTS We thank the medical staff and nurses of the Divisione Universitaria di Ematologia and of the Blood Bank, S. Giovanni Hospital, Torino, Italy, for help and patient care. This work was supported in part by Consiglio Nazionale delle Ricerche, Rome, Italy (special project ACRO, grant # PF39 to T.C. and # PF39 to A.P.) and by Associazione Italiana Ricerca sul Cancro, Milano, Italy. REFERENCES 1 Coiffier B. Towards a cure in indolent lymphoproliferative diseases? Eur J Cancer 1995;31a: Gribben JG, Freedman AS, Neuberg D et al. Immunologic purging of marrow assessed by PCR before ABMT for B-cell lymphoma. N Engl J Med 1991;325: Rohatiner A, Johnson P, Price C et al. Myeloablative therapy with autologous bone marrow transplantation as consolidation therapy for recurrent follicular lymphoma. J Clin Oncol 1994;12: Provan D, Bartlett-Pandite L, Zwicky C et al. 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