Introduction

High-dose chemotherapy (HDC) and bone marrow transplant (BMT) and, in recent years, autologous peripheral blood stem cell transplant (ASCT) are well recognized treatment options in diffuse large cell lymphoma (DLCL) and Hodgkin's lymphoma (HD) in a variety of settings. Chemotherapy¹ and growth factors (G-CSF, GM-CSF) have demonstrated their capacity to increase the circulating population of peripheral blood stem cell (PBSC) many-fold.^{2, 3, 4} Combining the two approaches has a synergistic effect.⁵ ASCT is superior to BMT in terms of quicker recovery from myelosuppression, easier to perform, cheaper and potentially less hazardous.^{6, 7, 8, 9} Many mobilization regimens have been reported in the literature but cyclophosphamide (1–7 g/m²) followed by G-CSF is one of the most commonly reported in patients with lymphoma.^{10, 11, 12, 13, 14, 15, 16, 17} Lately, other combinations with superior antitumor efficacy have been tried with success.^{13, 15, 18, 19, 20, 21, 22}

ESHAP²³ (Etoposide 40 mg/m² i.v. over 1–3 h, daily for 4 days, solumedrol 500 mg i.v. daily for 4 days, cisplatin 25 mg/m²/day i.v. as a continuous infusion over 24 h daily for 4 days and Ara-C 2 Gm/m² i.v. over 2–3 h, following completion of Cisplatin on day 5) is a well accepted salvage chemotherapy for patients with DLCL and HD. Limited data is available for ESHAP chemotherapy as a mobilizer. Few available studies had shown that ESHAP can be used safely as a mobilizer regimen.^{19, 20, 22}

Materials and methods

Patients

All patients with relapsed or refractory DLCL and HD who had HDC ASCT were identified by the Oncology Data Unit. From 1996 to November 2004, 131 patients with relapsed or refractory DLCL and HD who had received either ESHAP alone or ESHAP alternating with some other salvage chemotherapy regimen such as IMVP-16, IMCCNU, MIME, but ESHAP as mobilization chemotherapy prior to planned ASCT were selected for this analysis. All patients were admitted to the hospital for chemotherapy infusion. Responding DLCL patients and responding or stable HD patients (WHO response criteria) received ESHAP for mobilization of PBSC. Four patients did not mobilize and were excluded from this analysis. These patients had either cyclophosphamide for mobilization or bone marrow harvest for stem cell collection. A total of 127 patients, who had HDC ASCT are included in this analysis. Patients received fixed dose G-CSF (neupogen, Roche Oncology) 300 g SC twice a day starting 24–36 h after finishing mobilizing ESHAP and continued until the morning of last apheresis. Carmustine, etoposide, cytarabine and melphalan (BEAM) was used as HDC. Stem cell reinfusion was performed on day 0 and G-CSF 300 g SC twice a day was started from day +1 till absolute neutrophil count (ANC) reached 0.5 10⁹/l for 3 days or 1000 10⁹/l for 1 day in most cases.

Apheresis procedure

Daily apheresis using the COBE Spectra BCT, to collect a minimum of 2 10⁶ CD34+ cells/kg body weight (ideally 5 10⁶ CD34+ cells/kg body weight), was initiated when peripheral blood CD34+cell count exceeded 20 CD34+ cells/l. First apheresis volume was 10 l (patients 70 kg), 12 l (70–95 kg), and 15 l (>95 kg). Patients <60 kg often had <9 l due to tolerability. The apheresis product was mixed with the same volume of minimal essential medium containing 20% dimethylsulfoxide and stored at -196°C. Product volume, CFU-GM, CFU-GMME and CFU-E (BFUE) were also recorded.

CD34 enumeration

CD34 enumeration was performed on aliquots of apheresis samples using a single-platform flow cytometric method based on ISHAGE gating strategy as previously described.²⁴ Briefly, 50 l of diluted cells were incubated in TRUECOUNT™ tubes (BD Biosciences, #340334) containing fluorescence-labeled reference beads with antibodies against CD45-FITC (BD # 347463)/CD34-PE (clone 8G12, BD # 348057) and a viability dye 7-amino actinomycin D (7-AAD) (Coulter # IM-3422). Cells will be lysed with NH₄Cl. Samples were analyzed on a FACScalibur flow cytometer (BD Biosciences, USA) with a 488-nm argon laser and Cell Quest 3.1 software (BD Biosciences, USA). The number of CD34+ cells per microliter was calculated using the following formula:

CFU-GM, CFU-GMME and CFU-E assays

The assay for CFU-Mix, BFU-E and CFU-GM was carried out as described elsewhere.²⁵ Briefly, MNC were plated in duplicate in MethoCult™ H4433 and H4534 media (Stem Cell Technologies, Vancouver, Canada) at a concentration of 10 10⁴/ml. Cultures were incubated at 37°C and in 5% CO₂ for 9–14 days, colonies were then scored directly under an inverted microscope and classified as burst-forming units erythroid (BFU-E), CFU-GM and CFU-GMME. A CFU-GM was defined on the basis of its ability to produce at least 40 cells, and CFU-GMME should contain at least 20 cells of GM lineage in close proximity to the erythroid component.

Engraftment evaluation

Engraftment was defined as the first day when ANC of 0.5 10⁹/l was reached (lasting for at least 3 days) after the initial nadir post HDC ASCT. Various definitions are used to describe platelet engraftment; we used the International Bone Marrow Transplant Registry (IBMTR) definition, that is the first day of three consecutive complete blood counts with platelets 20 10⁹/l after the initial nadir post HDC ASCT in the absence of platelet transfusion or day 7 post last platelet transfusion (platelet transfusion +7 ), provided that the platelet counts on day +7 post transfusion remained 20 10⁹/l. We have also reported the more practically used platelet engraftment definition that is last platelet transfusion +1 day provided that the platelet counts on that day remained 20 10⁹/l. Many patients were discharged home and came back to our clinic 5–10 days after last platelet transfusion and at that time platelets were checked. Platelet transfusion threshold was 10 10⁹/l for asymptomatic patients and for febrile patients it was <20 10⁹/l. For few patients with deep venous thrombosis who were receiving heparin infusion, platelets were kept >30 10⁹/l.

Statistical analysis

SPSS Version 10 was used for statistical analyses. Pearson's correlation (continuous variables) or one-way analyses of variance (ANOVA) (categorical variables) were utilized to determine the significant associations with CD34+ cells collection. Pairwise comparisons were performed if ANOVA or ANCOVA were significant at the 0.05 level. Univariate regression models was created to investigate the effect of histology and weight on CD34+ cells collection. Results were considered significant if P<0.05 (two-sided).

Results

A total of 131 consecutive patients, who received ESHAP chemotherapy for mobilization followed by HDC ASCT were included in this analysis. Four patients failed mobilization and were excluded from the analysis. In all, 127 patients with successful mobilization are included in this analysis. Patient's characteristics are shown in Table 1. The distribution of total number of cycles is shown in Table 1 for patients with DLCL and HD. For patients who receiving CHOP (42 patients) as initial treatment, the distribution was <6 cycles (six patients), six cycles (23 patients), seven cycles (two patients) and eight cycles (11 patients). For patients who received ABVD (48 patients), it was <6 cycles (seven patients), six cycles (22 patients), seven cycles (four patients) and eight cycles (15 patients). Patients who received 'others' have mostly MOPP or COPP or MOPP/ABV chemotherapy, <6 cycles (six patients), six cycles (15 patients), seven cycles (two patients), eight cycles (five patients) and >8 (seven patients).

Table 1 - Patient's characteristics.

Full table

The dose of G-CSF was 300 g SC twice a day starting 24–36 h after finishing mobilizing ESHAP in all but three patients (480, 200 and 230 g SC twice a day).

Autologous stem cell collection

The total number of aphereses performed was 171 in 127 patients. The median day of apheresis from the first day of starting mobilization ESHAP was 14 days (range 12–21 days). This was day 12 in 8%, day 13 in 26%, day 14 in 35%, day 15 in 15%, day 16 in 7% and day 17 to 21 in the remaining 8%. Only one patient was collected on day 21. Median total CD34+ cells/kg collected were 6.9 10⁶ (DLCL 5.17 10⁶ and HD 7.6 10⁶). Patients requiring only one apheresis (93 patients) had CD34+ cells collection of 8.6 10⁶/kg and those requiring >1 apheresis (34 patients) had CD34+ cells collection of 4.5 10⁶ (P=0.001). Results of apheresis, CD34+ cells collection and CFU-GM, CFU-GMME and CFU-E (BFUE) are also shown in Table 2.

Table 2 - Total apheresis product.

Full table

Weight of patients and CD34+ cells collection

As the dose of G-CSF used in this group is different to the conventional weight based dosing, CD34+ cells collection results for different weight groups and histology are also presented in Tables 3, 4 and 5.

Table 3 - Total CD34+cell collection according to histology and weight.

Full table

Table 4 - Total CD34+cell collection in DLCL patients only.

Full table

Table 5 - Total CD34+cell collection in HD patients only.

Full table

Engraftment data

Neutrophil engraftment was 100%. Four patients failed platelet engraftment (platelets <20 10⁹/l by day 30 without transfusion or 20 10⁹/l with transfusion only). Of these, one patient died on day 61 due to treatment related mortality (septic shock post transplant), one patient received the last platelet transfusion on day +24 and engrafted on day 31 as per IBMTR definition, one patient with four previous lines of chemo and more than 20 prior chemotherapy cycles engrafted after 2 months and the fourth patient had progressive disease post HDC ASCT and failed engraftment. This is shown in Table 6.

Table 6 - Engraftment data.

Full table

Discussion

The optimal way to mobilize stem cells is still under investigation. For patients with non-Hodgkin's lymphoma (NHL) and HD undergoing HDC ASCT, the current standard approach is the use of chemotherapy and G-CSF as a mobilization regimen. Cyclophosphamide (1–7 g/m²) is one of the most common single agent chemotherapy reported for mobilization. Other combinations, such as ESHAP, DHAP and ifosfamide based salvage chemotherapy with superior antitumor activity are also tried with good success.^{13, 15, 18, 19, 20, 21, 22} There is a wide range of doses and schedules of G-CSF and other cytokines described in the literature for mobilization.^{7, 26, 27, 28} Currently the most common is G-CSF 5–10 g SC/kg/day.

Stem cell collection may be influenced by multiple factors such as type of lymphoma, age, stage, type and frequency of previous chemotherapy, radiation therapy (XRT), mobilization regimen and type of apheresis machine.^{29, 30, 31, 32, 33, 34} Most reports had a very diverse group of patients including HD and low, intermediate and high-grade NHL, acute myelogenous leukemia and even some solid cancers. These patients have diverse characteristics with a wide range of variables. They often had very different pre-salvage chemotherapy exposure and a variety of salvage chemotherapies due to the different nature of their disease. This makes it very difficult to draw a conclusion for a specific disease and CD34+ cell collection in that disease.

Our data is the largest single institution retrospective data to report the use of ESHAP as mobilization regimen in a very uniform set of patients. Unlike older studies where patients are offered HDC ASCT after multiple chemotherapy regimen failure and extensive pretransplant chemotherapy exposure, our patients reflect the recent trends in DLCL and HD therapy that is, early transplant for relapsed and refractory patients. Only 20 patients (18 with HD) in our study had >8 cycles of chemotherapy prior to the start of mobilization regimen. There is no low grade/indolent/mantle cell NHL included in our analysis. Our patients have a median age of 26 years at transplant. This is simply a reflection of age distribution of DLCL (median age 48 years for DLCL and 45 years for all NHL) and HD (median age 19 years) in our population. 65% of DLCL and 70% of HD patients are younger than 60 years in this country (recent published data from National Cancer Registry of Kingdom of Saudi Arabia).

Three other studies^{19, 20, 35} have also reported the use of ESHAP as a mobilization regimen in NHL and HD. It is difficult to compare our results with other reports due to the difference in patient population, type and dosing of growth factors, stem cell collection technique and the machine used. Petit¹⁹ reported median CD34+ cell collection of 7.4 10⁶/kg in 22 patients and Watts²⁰ reported median CD34+ cell collection of 4.8 10⁶/kg in 78 patients. Both ours and Watts²⁰ study showed that >70% patients required only one apheresis. Lee et al.³⁵ described 26 patients with different types of NHL. All the patients had minimum of two aphereses and the median CD34+ cells collected were 17.1 10⁶/kg. Although 81% of these patients had 5 10⁶/kg CD34+ cells collected after the first apheresis, all these patients underwent second apheresis. Our data shows the same result as Watts et al. and Lee et al., that 5 10⁶/kg CD34+ cells are collected in >70% patients with one apheresis.

Different institutions have different guidelines for the starting time of G-CSF after a specific chemotherapy regimen. The minimum acceptable dose and the duration of G-CSF in this setting in not known. We used fixed dose of G-CSF 300 g SC twice a day starting 24–36 h after finishing mobilizing ESHAP rather than the standard weight based dosing of 5–10 g/kg/day. Given the wide range of G-CSF dosing described in the literature⁷ and an appropriate CD34+ cells collection in almost all cases, this fixed dose falls in the middle of different dosing schedules. Given the wide range of equally good dosing, our fixed dose was able to produce a good result even in patients >70 kg. At the same time, it also indicates that most likely lower doses of G-CSF can produce the same results in the same setting.

Our data clearly and convincingly demonstrate that ESHAP+G-CSF 300 g SC twice a day starting 24–36 h after finishing mobilizing ESHAP for autologous peripheral blood stem cell mobilization in patients with relapsed or refractory DLCL and HD is appropriate. Fixed dose G-CSF can safely replace weight-based dosing regimen in this setting. Other appropriate dosing and timing of G-CSF in this setting will require more investigation in a uniform set of patients.

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Acknowledgements

We appreciate the assistance of Aneta Mclean from the apheresis lab and Ibrahim Abdullah Al-Hassan for statistical analysis.